Non-human animals with modified immunoglobulin heavy chain sequences

ABSTRACT

Non-human animals, e.g., mammals, e.g., mice or rats, are provided comprising an immunoglobulin heavy chain locus that comprises a rearranged human immunoglobulin heavy chain variable region nucleotide sequence. The rearranged human immunoglobulin heavy chain variable region nucleotide sequence may be operably linked to a heavy or light chain constant region nucleic acid sequence. Also described are genetically modified non-human animals comprising an immunoglobulin light chain locus comprising one or more but less than the wild type number of human immunoglobulin light chain variable region gene segments, which may be operably linked to a light chain constant region nucleic acid sequence. Also provided are methods for obtaining nucleic acid sequences that encode immunoglobulin light chain variable domains capable of binding an antigen in the absence of a heavy chain.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of priority to U.S. Provisional PatentApplication No. 61/766,765, filed Feb. 20, 2013, and to U.S. ProvisionalPatent Application No. 61/879,338, filed Sep. 18, 2013, which are herebyincorporated by reference in their entireties.

SEQUENCE LISTING

In accordance with 37 CFR § 1.52(e)(5), a Sequence Listing in the formof a text file (entitled “1270US_SL.txt,” created on Feb. 20, 2014, andis 87,000 bytes in size) is incorporated herein by reference in itsentirety.

FIELD OF THE INVENTION

Genetically modified non-human animals, e.g., rodents such as mice andrats, comprising a rearranged human heavy chain variable region nucleicacid sequence (i.e., a rearranged heavy chain VDJ sequence) operablylinked to a constant region nucleic acid sequence. In some embodiments,the animals are genetically engineered to have an immunoglobulin locuscomprising a rearranged heavy chain variable region (a VDJ sequence)nucleic acid sequence operably linked to an immunoglobulin constantregion gene sequence, wherein the VDJ sequence is a human VDJ sequence,and the constant region gene sequence is human or non-human. In someembodiments, the non-human animals containing a genetically modifiedimmunoglobulin locus comprise: (1) a first nucleotide sequence thatencodes a rearranged heavy chain variable domain (i.e., where the firstnucleotide sequence is a rearranged human immunoglobulin heavy chainvariable region nucleotide sequence), wherein the first nucleotidesequence is operably linked to a light chain (e.g., a κ or λ lightchain) constant region gene sequence; and (2) a second nucleotidesequence that encodes a human or non-human light chain (e.g., a κ or λlight chain) variable domain (i.e., where the second nucleotide sequenceis an unrearranged human immunoglobulin light chain variable nucleotidesequence), wherein the second nucleotide sequence is operably linked toa heavy chain constant region gene sequence. In some embodiments, thenon-human animals comprise a genetically modified immunoglobulin heavychain locus comprising a rearranged human immunoglobulin heavy chainvariable region nucleotide sequence, wherein the rearranged heavy chainvariable domain comprises a heavy chain V gene segment (V_(H)) sequenceoperably linked, via a spacer, to a heavy chain J gene segment (J_(H))sequence, and wherein the spacer comprises at least one amino acidresidue. Genetically modified non-human animals, e.g., rodents such asmice and rats, are provided comprising in their genomes: (i) arearranged human heavy chain variable region nucleic acid sequenceoperably linked to a constant region nucleic acid sequence; and (ii) animmunoglobulin light chain locus comprising one or more but less thanthe wild type number of human light chain variable region gene segments.Genetically modified non-human animals are provided comprising in theirgenomes: (i) a rearranged human heavy chain variable region nucleic acidsequence operably linked to a constant region nucleic acid sequence; and(ii) an immunoglobulin light chain locus comprising one or more but lessthan the wild type number of human immunoglobulin light chain variableregion gene segments. In some embodiments, at least one of the variableregion gene segments encodes one or more histidine residues that is/arenot encoded by a corresponding human germline light chain variableregion gene segment. Methods of making the genetically modifiednon-human animals described herein are provided. Methods for producingimmunoglobulin light chain (e.g., a κ or λ light chain) variable regionsequences that can bind an antigen in the absence of a heavy chain,and/or can be associated with a rearranged heavy chain variable domainand/or exhibit pH-dependent antigen binding characteristics, areprovided, which are useful for producing bispecific antibodies.

BACKGROUND

Bispecific antibodies are multifunctional antibodies that compriseantigen-binding sites that can bind two distinct antigenic determinantsand have emerged as one of the major therapeutic biologics for treatingmany diseases, including cancer. While a variety of bispecificantibodies with dual antigen-binding properties have been developedrecently, the specificity and affinity of immunoglobulin light chain orheavy chain variable domains in the conventional bispecific antibodieshad to be sacrificed to some extent because, in the conventionalbispecific antibodies, either only a heavy chain or a light chainvariable domain contributes to binding to each antigenic determinant,whereas, in regular antibodies, both light and heavy chain variableregions can contribute to binding to the same antigenic determinant. Inaddition, in achieving a desirable level of efficacy, therapeuticantibodies, e.g., bispecific therapeutic antibodies, often require highor multiple doses of antibodies due to their limited recyclability invivo.

Most antigen-binding proteins that target two antigens or epitopesdeveloped so far comprise two antigen-binding arms: (i) a firstantigen-binding arm comprising an immunoglobulin heavy-light chainvariable domain pair that contributes to binding to a first antigen orepitope; and (ii) a second antigen-binding arm comprising a secondheavy-light chain variable domain pair that contributes to binding to asecond antigen or epitope. These antigen-binding proteins, thoughbispecific in the context of the whole antigen-binding protein, are notnecessarily bispecific within each antigen-binding arm, limiting the useof the antigen-binding proteins in multi-specific formats, e.g.,tri-specific antigen-binding proteins. As disclosed herein, a non-humananimal that expresses a universal heavy chain variable domain may beemployed as a general tool for making antigen-binding proteins for usein many different formats of antigen-binding proteins.

SUMMARY

There is a need in the art to generate immunoglobulin light chainvariable domain sequences in which antigen specificity and affinityresults solely or primarily from, and/or resides solely or primarily in,immunoglobulin light chain variable domain diversity. Such sequenceswould be extremely useful in designing antigen-binding proteins, e.g.,bispecific antibodies, in which each variable domain is separatelyresponsible for distinct antigen-specific binding. Various aspects andembodiments described herein are based in part on the surprisingdiscovery that genetically modified non-human animals comprisingimmunoglobulin heavy chain variable domains encoded by a rearrangedheavy chain variable gene sequence (e.g., a rearranged heavy chain VDJsequence) can meet this need. Non-human animals encoding a rearrangedimmunoglobulin heavy chain variable domain (i.e., a universal heavychain variable domain) focus the mechanisms of antibody diversificationon unrearranged (i.e., diversifiable) antibody light chain variabledomain(s). Non-human animals include, e.g., mammals and, in particularembodiments, rodents (e.g., mice, rats, or hamsters).

Genetically modified non-human animals are provided that, uponstimulation with an antigen of interest, produce antibodies withantigen-binding specificity residing solely or primarily in the antibodylight chain variable domains. Light chain antibody variable domain aminoacids and corresponding nucleic acid sequences can be identified fromantibodies produced by such genetically modified animals, and thesequences can be utilized in recombinant antibodies or otherantigen-binding proteins to develop light chain variable domains thatbind an antigenic determinant independently (and with sufficientspecificity and affinity) from heavy chain variable domains. Moreover,the utility of genetically modified animals comprising a rearrangedheavy chain variable domain (i.e., comprising a prearranged heavy chainvariable domain gene sequence) can be applied by placing a nucleotidesequence encoding the rearranged heavy chain variable domain in avariety of genomic contexts, e.g., in different immunoglobulin loci.Rearranged heavy chain variable domain gene sequences can be targeted toa heavy chain locus or a light chain locus such that the rearrangedheavy chain variable domain sequences can be operably linked to a heavyor light chain constant sequence, either human or non-human. Rearrangedheavy chain variable domain gene sequences can be placed anywhere in thegenome in operable linkage with human, non-human, or mixedhuman/non-human immunoglobulin constant region sequences. Furthermore,non-human animals comprising a nucleotide sequence encoding a rearrangedheavy chain variable domain can be combined with additional geneticmodifications of immunoglobulin loci (e.g., crossbred to animalscomprising additional genetic modifications of immunoglobulin loci). Forexample, the focused diversification imparted by a rearranged heavychain variable domain gene sequence targeted to a light chain locus canbe paired with a light chain variable domain gene sequence inserted intoa heavy chain locus, thereby generating animals that fully utilize thetiming and diversification of a genomic context of choice (e.g., thediversification mechanisms of the heavy chain locus) to increasediversity of antibody variable gene sequence of choice (e.g., antibodylight chain variable gene sequences). In addition, by utilizing micethat have a restricted (limited) light chain variable region genesegment repertoire (e.g., a restricted number of light chain variablegene sequences that comprise one or more but less than the wild typenumber of human V_(L) gene segments in combination with the singlerearranged heavy chain sequence described above), an immunoglobulinlight chain variable domain that can more efficiently pair with animmunoglobulin heavy chain variable domain can be produced.

Thus, genetically modified non-human animals (e.g., rodents such asmice, rats, or hamsters) are provided that comprise an immunoglobulinlocus comprising a rearranged human immunoglobulin heavy chain variableregion (i.e., a nucleotide sequence that encodes a rearranged heavychain variable domain; i.e., a rearranged heavy chain VDJ sequence).

In various aspects, the only genomic heavy chain variabledomain-encoding nucleic acid sequence expressed by the geneticallymodified non-human animals is the rearranged heavy chain variabledomain. Accordingly, the diversity of antibody heavy chain variabledomains produced by the genetically modified non-human animals isextremely restricted.

In some embodiments, genetically modified non-human animals are providedthat have in their genome an immunoglobulin locus that has beengenetically modified so that its variable region sequences consistessentially of a single rearranged human heavy chain variable region. Itis understood that different cells in such genetically modifiednon-human animals may not always have completely identical sequences inthe single rearranged human heavy chain variable region (e.g., due toreplication errors, somatic hypermutation, or other mechanisms), butregardless, such genetically modified non-human animals showdramatically restricted diversity of antibody heavy chain variabledomains as compared with animals having unrearranged heavy chainvariable sequences, and/or animals whose genomes include multiple heavychain variable region gene segments (e.g., multiple V, D, and/or Jsegments, particularly if unrearranged).

In various aspects, a genetically modified immunoglobulin heavy chainlocus is provided comprising a rearranged human immunoglobulin heavychain variable region nucleotide sequence (i.e., comprising a nucleotidesequence that encodes a rearranged heavy chain variable domain). Invarious aspects, the rearranged human immunoglobulin heavy chainvariable region nucleotide sequence and the rearranged heavy chainvariable domain it encodes are derived from a human V, D, and J genesegment. In various aspects, the rearranged human immunoglobulin heavychain variable region nucleotide sequence and the rearranged heavy chainvariable domain it encodes are derived from a human V_(H) gene and ahuman J_(H) segment. In various aspects, the rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence isoperably linked to a heavy chain constant region region gene sequence.In various aspects, the rearranged human immunoglobulin heavy chainvariable region nucleotide sequence is operably linked to a light chainconstant region region gene sequence. In various aspects, thegenetically modified immunoglobulin locus is present in the germline ofa non-human animal. In various aspects, the genetically modifiednon-human animals comprise the full complement of unrearranged lightchain variable gene segments capable of rearranging to form a lightchain gene in operable linkage with a light chain constant region genesequence. In other aspects, the genetically modified non-human animalscomprise a plurality but less than a full complement (i.e., less than awild type number) of unrearranged light chain variable gene segments. Invarious aspects, the unrearranged light chain variable gene segments areoperably linked to a heavy chain constant region gene sequence. Inspecific aspects, the non-human animal is a rodent, e.g., a mouse, rat,or hamster. In another aspect, a nucleic acid construct is providedcomprising a rearranged human immunoglobulin heavy chain variable region(i.e., comprising a nucleotide sequence that encodes a rearranged heavychain variable domain; i.e., a pre-rearranged heavy chain VDJ sequence)as described herein.

Numerous variations of genetically modified non-human animals with animmunoglobulin locus comprising a rearranged human immunoglobulin heavychain variable region nucleotide sequence (i.e., with an immunoglobulinlocus comprising a nucleotide sequence that encodes a rearranged heavychain variable domain) are disclosed herein. Each variation has thecapability to focus the mechanisms of antibody diversification onimmunoglobulin light chain variable region nucleotide sequences.

In various aspects, a nucleotide sequence that encodes a rearrangedheavy chain variable domain (i.e., a heavy chain variable domain encodedby a rearranged human immunoglobulin heavy chain variable regionnucleotide sequence) is operably linked to a human or non-human heavychain constant region gene sequence (e.g., a heavy chain constant regiongene sequence that encodes an immunoglobulin isotype selected from IgM,IgD, IgA, IgE, IgG, and combinations thereof). For example, geneticallymodified non-human animals are provided comprising immunoglobulin lociin which: (a) a first nucleotide sequence encodes a rearranged heavychain variable domain (i.e., where the first nucleotide sequence is arearranged human immunoglobulin heavy chain variable region nucleotidesequence), wherein the first nucleotide sequence is operably linked to ahuman or non-human (or mixed human/non-human) heavy chain constantregion gene sequence; and (b) a second nucleotide sequence encodes alight chain variable domain (i.e., where the second nucleotide sequenceis an unrearranged human immunoglobulin light chain variable nucleotidesequence), wherein the second nucleotide sequence is operably linked toa human or non-human light chain constant region gene sequence.

In another aspect, modified non-human animals are provided in which theanimals comprise a rearranged nucleotide sequence that encodes a heavychain variable domain, wherein the heavy chain variable domain comprisesa heavy chain variable (V_(H)) sequence that is operably linked, via aspacer, to a heavy chain J segment (J_(H)) sequence, wherein the spacerencodes at least one amino acid residue.

In another aspect, a non-human animal is provided comprising agenetically modified immunoglobulin locus that comprises a rearrangedhuman immunoglobulin heavy chain variable region nucleotide sequence(i.e., comprise a nucleic acid sequence encoding a rearranged heavychain variable domain; i.e., a rearranged heavy chain VDJ sequence),wherein the genetically modified immunoglobulin locus is present in thegermline of the non-human animal. In some embodiments, the geneticallymodified immunoglobulin locus is a heavy chain locus. In someembodiments, the genetically modified immunoglobulin locus is a lightchain locus

In another aspect, genetically modified non-human animals (e.g., rodentssuch as mice, rats, or hamsters) are provided having a geneticallymodified immunoglobulin genomic locus a rearranged human immunoglobulinheavy chain variable region nucleotide sequence, wherein the nucleotidesequence is operably linked to a human or non-human light chain (e.g., Kor A light chain) constant region gene sequence.

In another aspect, a non-human animal comprising a genetically modifiedimmunoglobulin locus is provided comprising: (a) a rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence operablylinked to a light chain constant region gene sequence; and (b) aunrearranged human or non-human light chain (e.g., κ or λ light chain)variable region nucleotide sequence operably linked to a human ornon-human heavy chain constant region gene sequence (e.g., a heavy chainconstant region gene sequence that encodes an immunoglobulin isotypeselected from IgM, IgD, IgA, IgE, IgG, and a combination thereof).

In another aspect, a genetically modified non-human animal is providedwith an immunoglobulin locus comprising:

-   -   (a) a first allele comprising:        -   (i) a first nucleotide sequence that encodes a rearranged            heavy chain variable domain (i.e., where the first            nucleotide sequence is a rearranged human immunoglobulin            heavy chain variable region nucleotide sequence) operably            linked to a heavy chain constant region gene sequence, and        -   (ii) a second nucleotide sequence that encodes a light chain            variable domain (i.e., where the second nucleotide sequence            is an unrearranged human immunoglobulin light chain variable            nucleotide sequence) operably linked to a light chain            constant region gene sequence; and    -   (b) a second allele comprising        -   (i) a third nucleotide sequence that encodes a light chain            variable domain (i.e., where the third nucleotide sequence            is an unrearranged human immunoglobulin light chain variable            nucleotide sequence) operably linked to a heavy chain            constant region gene sequence, and        -   (ii) a fourth nucleotide sequence that encodes the            rearranged heavy chain variable domain (i.e., where the            fourth nucleotide sequence is a rearranged human            immunoglobulin heavy chain variable region nucleotide            sequence) operably linked to a light chain constant region            gene sequence.

In various aspects, genetically modified non-human animals withunrearranged light chain variable region gene sequences or loci areprovided. In some embodiments, the genetically modified non-humananimals comprise a wild type number (i.e., all or substantially all) ofhuman immunoglobulin light chain variable region gene segments (i.e.,sequences). In other aspects, the non-human animals described hereincomprises a limited repertoire of light chain variable gene segments,e.g., (i) one, two or more but less than the wild type number of humanV_(L) gene segments; and (ii) one or more human J_(L) gene segments,operably linked to a non-human light chain constant region nucleic acidsequence. The heavy chain nucleic acid sequence and/or the light chainsegments may be present, e.g., in a transgene or at an endogenousimmunoglobulin locus.

In various aspects, genetically modified non-human animals are provided,wherein all immunoglobulin heavy chain variable domains of the animalare derived from the same rearranged variable heavy chain gene sequence,and wherein said variable domains are expressed cognate with a lightchain variable domain derived from one of at least one, two, or three ormore V_(L) gene segments and at least one, two, or three or more J_(L)gene segments. Additionally, genetically modified non-human animals(e.g., rodents, such as mice and rats) are provided comprising in theirgenomes: (i) an immunoglobulin heavy chain locus that comprises arearranged human heavy chain variable region nucleic acid sequenceoperably linked to a human or non-human heavy chain constant regionnucleic acid sequence; and (ii) an immunoglobulin light chain locuscomprising one or more but less than the wild type number of humanimmunoglobulin light chain variable region gene segments (e.g., twohuman V_(κ) gene segments and one or more human J_(κ) gene segments),operably linked to a human or non-human light chain constant regionnucleic acid sequence. In some embodiments, the light chain constantregion is a rat or a mouse constant region, e.g., a rat or a mouse Cκconstant region.

In another aspect, the genetically modified non-human animals asdescribed herein, upon stimulation with an antigen of interest, expressan antigen-binding protein comprising an immunoglobulin heavy chain anda light chain amino acid sequence, wherein the heavy chain amino acidsequence is derived from a genetically modified heavy chain locuscomprising a rearranged human heavy chain variable region nucleic acidsequence operably linked to a heavy chain constant region nucleic acidsequence. In certain aspects, the light chain amino acid sequence isderived from a genetically modified immunoglobulin light chain locuscomprising one or more but less than the wild type number of human V_(L)gene segments and (ii) two or more human J_(L) gene segments, operablylinked to a non-human light chain constant region nucleic acid sequence.

Genetically modified non-human animals are provided comprising in theirgenomes a rearranged human immunoglobulin heavy chain variable regionnucleic acid sequence that comprises a heavy chain V gene segment(V_(H)) that is operably linked, via a spacer, to a heavy chain J genesegment (J_(H)) sequence, wherein the spacer encodes at least one aminoacid (e.g., 2 amino acids, 3 amino acids, or 4 amino acids) and/or amodified D gene segment. In various embodiments, the rearranged heavychain variable region nucleic acid sequence is operably linked to ahuman or non-human heavy chain constant region nucleic acid sequence. Invarious embodiments, the non-human animals further comprise in theirgenomes a genetically modified immunoglobulin light chain locuscomprising one or more but less than the wild type number of humanimmunoglobulin light chain variable region gene segments, e.g., twohuman V_(κ) gene segments and one or more human J_(κ) gene segments,operably linked to a human or non-human light chain constant regionnucleic acid sequence.

Methods of making and using the genetically modified non-human animalsdescribed herein are also provided. Methods are provided for placing arearranged human heavy chain variable region nucleic acid sequence inoperable linkage with an immunoglobulin heavy or light chain constantregion nucleic acid sequence in the genome of a non-human animal.

In another aspect, methods are provided for obtaining an immunoglobulinlight chain variable region (V_(L)) amino acid sequence capable ofbinding an antigen independently from a heavy chain variable regionamino acid sequence.

In another aspect, a genetically modified immunoglobulin locusobtainable by any of the methods as described herein is provided.

In various aspects, antigen-binding proteins (e.g., antibodies) producedby or derived from the genetically modified non-human animals describedherein are provided. Also provided are methods for makingantigen-binding proteins, including multispecific (e.g., bispecific ortrispecific) antigen-binding proteins. Also provided are methods formaking an effector light chain immunoglobulin variable domains.

In another aspect, a pluripotent cell, induced pluripotent, ortotipotent stem cells derived from a non-human animal comprising thevarious genomic modifications described herein are provided. Cells thatcomprise a nucleus containing a genetic modification as described hereinare also provided, e.g., a modification introduced into a cell bypronuclear injection.

In various aspects, a non-human animal embryo comprising a cell whosegenome comprises an immunoglobulin heavy chain locus comprising arearranged human heavy chain variable region nucleic acid sequenceoperably linked to a constant region nucleic acid sequence is provided.In certain aspects, the non-human animal embryo further comprises animmunoglobulin light chain locus comprising two or more but less thanthe wild type number of human immunoglobulin light chain variable regiongene segments, operably linked to a light chain constant region nucleicacid sequence.

Also provided are methods for making nucleic acid sequences that encodean immunoglobulin light chain variable region (V_(L)) amino acidsequence capable of binding an antigen or an epitope thereofindependently from a heavy chain variable domain, comprising: (a)immunizing a non-human animal with an antigen of interest or animmunogen thereof, wherein the non-human animal comprises in its genome(i) a rearranged human immunoglobulin heavy chain variable regionnucleic acid sequence operably linked to a heavy chain constant regionnucleic acid sequence, and (ii) an unrearranged human immunoglobulinlight chain variable region nucleic acid sequence operably linked to alight chain constant region nucleic acid sequence; (b) allowing thenon-human animal to mount an immune response; (c) isolating from theimmunized non-human animal a cell comprising a nucleic acid sequencethat encodes a light chain variable domain that can bind the antigen;and (d) obtaining from the cell a nucleic acid sequence that encodes thelight chain variable domain (V_(L) domain) that can bind the antigen. Invarious embodiments, the cell is a lymphocyte, including, but notlimited to, natural killer cells, T cells, and B cells. In variousembodiments, the method further comprises (c)′ fusing the lymphocytewith a cancer cells, e.g., a myeloma cell.

Also provided are methods for making nucleic acid sequences that encodean immunoglobulin light chain variable region (V_(L)) amino acidsequence capable of binding an antigen or an epitope thereofindependently from a heavy chain variable domain, comprising: (a)immunizing a non-human animal with an antigen of interest or animmunogen thereof, wherein the non-human animal comprises in its genome(i) a rearranged human immunoglobulin heavy chain variable regionnucleic acid sequence operably linked to a heavy chain constant regionnucleic acid sequence, and (ii) two or more but less than the wild typenumber of human immunoglobulin light chain variable region gene segments(V_(L) and J_(L)) operably linked to a light chain constant regionnucleic acid sequence; (c) isolating from the immunized non-human animala cell comprising a nucleic acid sequence that encodes a light chainvariable domain that can bind the antigen; and (d) obtaining from thecell a nucleic acid sequence that encodes the light chain variabledomain (V_(L) domain) that can bind the antigen. In various embodiments,the cell is a lymphocyte, including, but not limited to, natural killercells, T cells, and B cells. In various embodiments, the method furthercomprises (c)′ fusing the lymphocyte with a cancer cells, e.g., amyeloma cell.

Also provided are methods for making nucleic acid sequences that encodean immunoglobulin light chain variable region (V_(L)) amino acidsequence capable of binding an antigen or an epitope thereofindependently from a heavy chain variable domain, comprising: (a)immunizing a non-human animal with an antigen of interest or animmunogen thereof, wherein the non-human animal comprises in its genome:(i) a rearranged human immunoglobulin heavy chain variable regionnucleic acid sequence operably linked to a light chain constant regionnucleic acid sequence, and (ii) human immunoglobulin light chainvariable region gene segments (V_(L) and J_(L)) operably linked to aheavy chain constant region nucleic acid sequence; (c) isolating fromthe immunized non-human animal a cell comprising a nucleic acid sequencethat encodes a light chain variable domain that can bind the antigen;and (d) obtaining from the cell a nucleic acid sequence that encodes thelight chain variable domain (V_(L) domain) that can bind the antigen. Invarious embodiments, the cell is a lymphocyte, including, but notlimited to, natural killer cells, T cells, and B cells. In variousembodiments, the method further comprises (c)′ fusing the lymphocytewith a cancer cells, e.g., a myeloma cell.

Also provided are methods for making antigen-binding proteins,comprising:

-   -   (a) immunizing a non-human animal with a first antigen that        comprises a first epitope or immunogenic portion thereof,        wherein the non-human animal comprises in its genome: (i) a        rearranged human immunoglobulin heavy chain variable region        nucleic acid sequence operably linked to a heavy chain constant        region nucleic acid sequence, and (ii) an unrearranged human        immunoglobulin light chain variable region nucleic acid sequence        operably linked to a light chain constant region nucleic acid        sequence;    -   (b) allowing the non-human animal to mount an immune response to        the first epitope or immunogenic portion thereof;    -   (c) isolating from the non-human animal a cell comprising a        nucleic acid sequence that encodes a light chain variable domain        that specifically binds the first epitope or immunogenic portion        thereof;    -   (d) obtaining from the cell of (c) the nucleic acid sequence        that encodes a light chain variable domain that specifically        binds the first epitope or immunogenic portion thereof;    -   (e) employing the nucleic acid sequence of (c) in an expression        construct, fused to a human immunoglobulin constant region        nucleic acid sequence; and    -   (f) expressing the nucleic acid sequence of (c) in a production        cell line that expresses a human immunoglobulin heavy chain that        specifically binds a second antigen or epitope thereof to form        an antigen-binding protein whose light chain is encoded by the        nucleic acid of (c) and that binds the first epitope or        immunogenic portion thereof independently from the heavy chain,        and whose heavy chain specifically binds the second antigen or        epitope.

Also provided are methods for making antigen-binding proteins,comprising:

-   -   (a) immunizing a non-human animal with a first antigen that        comprises a first epitope or immunogenic portion thereof,        wherein the non-human animal comprises in its genome: (i) a        rearranged human immunoglobulin heavy chain variable region        nucleic acid sequence operably linked to a heavy chain constant        region nucleic acid sequence, and (ii) two or more but less than        the wild type number of human immunoglobulin light chain        variable region gene segments (V_(L) and J_(L)) operably linked        to a light chain constant region nucleic acid sequence;    -   (b) allowing the non-human animal to mount an immune response to        the first epitope or immunogenic portion thereof;    -   (c) isolating from the non-human animal a cell comprising a        nucleic acid sequence that encodes a light chain variable domain        that specifically binds the first epitope or immunogenic portion        thereof;    -   (d) obtaining from the cell of (c) the nucleic acid sequence        that encodes a light chain variable domain that specifically        binds the first epitope or immunogenic portion thereof;    -   (e) employing the nucleic acid sequence of (c) in an expression        construct, fused to a human immunoglobulin constant region        nucleic acid sequence; and    -   (f) expressing the nucleic acid sequence of (c) in a production        cell line that expresses a human immunoglobulin heavy chain that        specifically binds a second antigen or epitope thereof to form        an antigen-binding protein whose light chain is encoded by the        nucleic acid of (c) and that binds the first epitope or        immunogenic portion thereof independently from the heavy chain,        and whose heavy chain specifically binds the second antigen or        epitope.

Also provided are methods for making antigen-binding proteins,comprising:

-   -   (a) immunizing a non-human animal with a first antigen that        comprises a first epitope or immunogenic portion thereof,        wherein the non-human animal comprises in its genome: (i) a        rearranged human immunoglobulin heavy chain variable region        nucleic acid sequence operably linked to a light chain constant        region nucleic acid sequence, and (ii) human immunoglobulin        light chain variable region gene segments (V_(L) and J_(L))        operably linked to a heavy chain constant region nucleic acid        sequence;    -   (b) allowing the non-human animal to mount an immune response to        the first epitope or immunogenic portion thereof;    -   (c) isolating from the non-human animal a cell comprising a        nucleic acid sequence that encodes a light chain variable domain        that specifically binds the first epitope or immunogenic portion        thereof;    -   (d) obtaining from the cell of (c) the nucleic acid sequence        that encodes a light chain variable domain that specifically        binds the first epitope or immunogenic portion thereof;    -   (e) employing the nucleic acid sequence of (c) in an expression        construct, fused to a human immunoglobulin constant region        nucleic acid sequence; and    -   (f) expressing the nucleic acid sequence of (c) in a production        cell line that expresses a human immunoglobulin heavy chain that        specifically binds a second antigen or epitope thereof to form        an antigen-binding protein whose light chain is encoded by the        nucleic acid of (c) and that binds the first epitope or        immunogenic portion thereof independently from the heavy chain,        and whose heavy chain specifically binds the second antigen or        epitope.

In various aspects, a non-human animal is provided comprising in itsgermline genome an immunoglobulin heavy chain locus that comprises arearranged human immunoglobulin heavy chain variable region nucleotidesequence. In some embodiments, the non-human animal is a mammal. In someembodiments, the mammal is a rodent. In some embodiments, the rodentselected from the group consisting of a mouse, a rat, and a hamster. Insome embodiments, the non-human animal is homozygous for the rearrangedhuman immunoglobulin heavy chain variable region nucleotide sequence. Insome embodiments, the rearranged human immunoglobulin heavy chainvariable region nucleotide sequence is operably linked to a non-humanheavy chain constant region gene sequence. In certain embodiments, thenon-human heavy chain constant region gene sequence encodes an Fc. Inparticular embodiments, the non-human heavy chain constant region genesequence is a mouse or a rat heavy chain constant region gene sequence.In some embodiments, the non-human animal is a rodent, and therearranged human immunoglobulin heavy chain variable region nucleotidesequence is operably linked to a human heavy chain constant region genesequence. In particular embodiments, the heavy chain constant regiongene sequence is selected from a C_(H)1, a hinge, a C_(H)2, a C_(H)3,and a combination thereof. In some embodiments, the rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence isderived from a human heavy chain V_(H) gene segment, a human heavy chainD gene segment, and a human heavy chain J_(H) gene segment. In certainembodiments, the rearranged human immunoglobulin heavy chain variableregion nucleotide sequence is derived from a human germline heavy chainV_(H) segment, a human germline heavy chain D segment, and a humangermline heavy chain J_(H) segment. In some embodiments, the rearrangedhuman immunoglobulin heavy chain variable region nucleotide sequenceencodes the sequence of human V_(H)3-23/GY/J_(H)4-4. In someembodiments, substantially all endogenous functional V_(H), D, and J_(H)gene segments are deleted from the immunoglobulin heavy chain locus ofthe non-human animal or rendered non-functional. In some embodiments,the non-human animal comprises a modification that deletes or rendersnon-functional endogenous functional V_(H), D, and J_(H) gene segments;and the non-human animal comprises the rearranged human immunoglobulinheavy chain variable region nucleotide sequence, wherein the rearrangedhuman immunoglobulin heavy chain variable region nucleotide sequence ispresent ectopically. In some embodiments, an immunoglobulin heavy chainvariable domain encoded by the rearranged heavy chain variable regionnucleotide sequence is not immunogenic to the non-human animal. In someembodiments, the non-human animal comprises an Adam6a gene, an Adam6bgene, or both. In some embodiments, the non-human animal furthercomprises a nucleotide sequence encoding an unrearranged humanimmunoglobulin light chain (V_(L)) gene segment and an unrearrangedhuman immunoglobulin light chain J gene segment. In certain embodiments,the nucleotide sequence encoding the unrearranged light chain V genesegment (V_(L)) and the unrearranged light chain (J_(L)) gene segment isoperably linked to an immunoglobulin light chain constant region genesequence. In particular embodiments, the light chain constant regiongene sequence is selected from a rodent and a human constant region genesequence. In yet more particular embodiments, the rodent is selectedfrom a mouse, a rat, and a hamster. In certain embodiments, theunrearranged human immunoglobulin light chain (V_(L)) gene segment andthe unrearranged human immunoglobulin (J_(L)) gene segment are operablylinked, at an endogenous rodent locus, to a rodent immunoglobulinconstant region gene sequence. In some embodiments, the immunoglobulinheavy chain locus comprises a plurality of copies of the rearrangedhuman immunoglobulin heavy chain variable region nucleotide sequence.

In additional aspects are provided a non-human immunoglobulin heavychain locus in a genome of a non-human germ cell comprising a rearrangedhuman immunoglobulin heavy chain variable region nucleotide sequenceoperably linked to a heavy chain constant region gene sequence, whereinthe constant region gene sequence comprises a non-human sequence, ahuman sequence, or a combination thereof. In some embodiments, therearranged human immunoglobulin heavy chain variable region nucleotidesequence is operably linked to an endogenous non-human immunoglobulinconstant region gene sequence. In certain embodiments, the endogenousnon-human immunoglobulin constant region gene sequence is a mouse or arat heavy chain constant region gene sequence.

In additional aspects, methods are provided for making a non-humananimal, the methods comprising:

-   -   (a) modifying a genome of a non-human animal to delete or render        non-functional endogenous functional immunoglobulin heavy chain        V_(H), D, and J_(H) gene segments; and    -   (b) placing in the genome a rearranged human immunoglobulin        heavy chain variable region nucleotide sequence.        In some embodiments, the rearranged human immunoglobulin heavy        chain variable region nucleotide sequence is operably linked to        a non-human immunoglobulin heavy chain constant region gene        sequence. In certain embodiments, the non-human immunoglobulin        heavy chain constant region gene sequence is a mouse or rat        immunoglobulin heavy chain constant region gene sequence. In        some embodiments, the rearranged human immunoglobulin heavy        chain variable region nucleotide sequence is placed at an        endogenous immunoglobulin heavy chain locus in the genome. In        some embodiments, the rearranged human immunoglobulin heavy        chain variable region nucleotide sequence is present in a        germline genome of the non-human animal. In some embodiments,        the rearranged human immunoglobulin heavy chain variable region        nucleotide sequence is present at an ectopic locus in the        genome. In some embodiments, the non-human animal comprises a        plurality of copies of the rearranged human immunoglobulin heavy        chain variable region nucleotide sequence. In some embodiments,        the non-human animal comprises an Adam6a gene, an Adam6b gene or        both. In some embodiments, the non-human animal is a rodent        selected from the group consisting of a mouse, a rat, or a        hamster. In some embodiments are provided a non-human animal        that is heterozygous for the immunoglobulin heavy chain locus as        described herein, wherein the non-human animal expresses the        rearranged human immunoglobulin heavy chain variable region        nucleotide sequence predominantly from the immunoglobulin heavy        chain locus.

In additional aspects are provided non-human animals comprising agenetically modified immunoglobulin locus comprising:

-   -   (a) a rearranged human immunoglobulin heavy chain variable        region nucleotide sequence that is operably linked to a light        chain constant region gene sequence; and    -   (b) an unrearranged human immunoglobulin light chain variable        region nucleotide sequence that is operably linked to a heavy        chain constant region gene sequence.

In some embodiments, the non-human animal is a mammal. In particularembodiments, the mammal is selected from the group consisting of amouse, a rat, and a hamster. In some embodiments, the rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence isoperably linked to a κ light chain constant region gene sequence. Insome embodiments, the rearranged human immunoglobulin heavy chainvariable region nucleotide sequence is operably linked to a λ lightchain constant region gene sequence. In some embodiments, the lightchain constant region gene sequence is a mouse or a rat light chainconstant region gene sequence. In some embodiments, the light chainconstant region gene sequence is a human light chain constant regiongene sequence. In some embodiments, the heavy chain constant region genesequence is a mouse or a rat heavy chain constant region gene sequence.In some embodiments, the heavy chain constant region gene sequence is ahuman heavy chain constant region gene sequence. In some embodiments,the heavy chain constant region gene sequence encodes a sequenceselected from a CH1, a hinge, a CH2, a CH3, and a combination thereof.In some embodiments, the unrearranged human immunoglobulin light chainvariable region nucleotide sequence comprises a human κ light chainvariable domain gene sequence. In some embodiments, the unrearrangedhuman immunoglobulin light chain variable region nucleotide sequencecomprises a human λ light chain variable domain gene sequence. In someembodiments, the rearranged human immunoglobulin heavy chain variableregion nucleotide sequence is derived from a human heavy chain V_(H)gene segment, a human heavy chain D gene segment, and a human heavychain J_(H) gene segment. In certain embodiments, the rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence isderived from a human germline heavy chain V_(H) segment, a humangermline heavy chain D segment, and a human germline heavy chain J_(H)segment. In certain embodiments, the human V_(H) gene segment isselected from the group consisting of V_(H)1-2, V_(H)1-3, V_(H)1-8,V_(H)1-18, V_(H)1-24, V_(H)1-45, V_(H)1-46, V_(H)1-58, V_(H)1-69,V_(H)2-5, V_(H)2-26, V_(H)2-70, V_(H)3-7, V_(H)3-9, V_(H)3-11,V_(H)3-13, V_(H)3-15, V_(H)3-16, V_(H)3-20, V_(H)3-21, V_(H)3-23,V_(H)3-30, V_(H)3-30-3, V_(H) 3-30-5, V_(H)3-33, V_(H)3-35, V_(H)3-38,V_(H)3-43, V_(H)3-48, V_(H)3-49, V_(H)3-53, V_(H)3-64, V_(H)3-66,V_(H)3-72, V_(H)3-73, V_(H)3-74, V_(H)4-4, V_(H)4-28, V_(H)4-30-1,V_(H)4-30-2, V_(H)4-30-4, V_(H)4- 31, V_(H)4-34, V_(H)4-39, V_(H)4-59,V_(H)4-61, V_(H)5-51, V_(H)6-1, V_(H)7-4-1, V_(H)7-81, and a polymorphicvariant thereof. In certain embodiments, the human D gene segment isselected from the group consisting of D1-1, D1-7, D1-14, D1-20, D1-26,D2-2, D2-8, D2-15, D2-21, D3-3, D3-9, D3-10, D3-16, D3-22, D4-4, D4-11,D4-17, D4-23, D5-12, D5-5, D5-18, D5-24, D6-6, D6-13, D6-19, D6-25,D7-27, and a polymorphic variant thereof. In certain embodiments, thehuman J_(H) gene segment is selected from the group consisting ofJ_(H)1, J_(H)2, J_(H)3, J_(H)4, J_(H)S, J_(H)6, and a polymorphicvariant thereof. In some embodiments, wherein the rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence encodesthe sequence of human V_(H)3-23/GY/J_(H)4-4 (SEQ ID NO: 137). In someembodiments, the genetically modified immunoglobulin locus is present inthe germline of the non-human animal. In some embodiments, substantiallyall endogenous functional V_(H), D, and J_(H) gene segments are deletedfrom the immunoglobulin heavy chain locus of the non-human animal orrendered non-functional. In some embodiments, the non-human animalcomprises a modification that deletes or renders non-functionalendogenous functional V_(H), D, and J_(H) gene segments; and thenon-human animal comprises the rearranged human immunoglobulin heavychain variable region nucleotide sequence that is operably linked to thelight chain constant region gene sequence, wherein the rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence ispresent at an ectopic locus in the genome. In some embodiments, thenon-human animal comprises an Adam6a gene, an Adam6b gene or both. Insome embodiments, a heavy chain variable domain encoded by therearranged human immunoglobulin heavy chain variable region nucleotidesequence is not immunogenic to the non-human animal. In someembodiments, the genetically modified immunoglobulin locus comprises aplurality of copies of the rearranged human immunoglobulin heavy chainvariable region nucleotide sequence that is operably linked to the lightchain constant region gene sequence.

Additional aspects provide an immunoglobulin locus in a germline genomeof a non-human animal comprising:

-   -   (1) a rearranged human immunoglobulin heavy chain variable        region nucleotide sequence that is operably linked to a light        chain constant region gene sequence, and    -   (2) an unrearranged human immunoglobulin light chain variable        region nucleotide sequence that is operably linked to a heavy        chain constant region gene sequence.

In some embodiments, the light chain constant region gene sequence is aκ light chain constant region gene sequence. In some embodiments, thelight chain constant region gene sequence is a λ light chain constantregion gene sequence. In some embodiments, the light chain constantregion gene sequence is a mouse or rat light chain constant region genesequence.

Additional aspects provide methods of making a non-human animal thatcomprise a modified immunoglobulin locus, the methods comprising:

-   -   (a) modifying a genome of a non-human animal to delete or render        non-functional:        -   (i) endogenous functional immunoglobulin heavy chain V, D,            and J gene segments, and        -   (ii) endogenous functional immunoglobulin light chain V and            J gene segments;    -   and    -   (b) placing in the genome:        -   (i) a rearranged human immunoglobulin heavy chain variable            region nucleotide sequence that is operably linked to a            light chain constant region gene sequence, and        -   (ii) an unrearranged human immunoglobulin light chain            variable region nucleotide sequence that is operably linked            to a heavy chain constant region gene sequence.

In some embodiments, the unrearranged human immunoglobulin light chainvariable region nucleotide sequence encodes a κ light chain variabledomain. In some embodiments, the unrearranged human immunoglobulin lightchain variable region nucleotide sequence encodes a λ light chainvariable domain. In some embodiments, the heavy chain constant regiongene sequence is a non-human immunoglobulin heavy chain constant regiongene sequence. In certain embodiments, the non-human immunoglobulinheavy chain constant region gene sequence is a mouse or a rat heavychain constant region gene sequence. In particular embodiments, theheavy chain constant region gene sequence encodes a sequence selectedfrom a CH1, a hinge, a CH2, a CH3, and a combination thereof. In someembodiments, the non-human animal is a rodent selected from the groupconsisting of a mouse, a rat, or a hamster. In some embodiments, themodified immunoglobulin locus is present in a germline genome of thenon-human animal. In some embodiments, the non-human animal comprises anAdam6a gene, an Adam6b gene or both.

Also provided are non-human animals comprising a modified immunoglobulinheavy chain locus that comprises a rearranged human immunoglobulin heavychain variable region nucleotide sequence comprising a heavy chain Vsegment (V_(H)) sequence that is operably linked, via a spacer, to aheavy chain J segment (J_(H)) sequence, wherein the spacer comprises atleast one amino acid residue. In some embodiments, the non-human animalis a rodent. In some embodiments, the rodent is selected from the groupconsisting of a mouse, a rat, and a hamster. In some embodiments, therearranged human immunoglobulin heavy chain variable region nucleotidesequence is operably linked to a non-human heavy chain constant regiongene sequence. In some embodiments, the non-human heavy chain constantregion gene sequence is a mouse or a rat constant region gene sequence.In some embodiments, the rearranged human immunoglobulin heavy chainvariable region nucleotide sequence is operably linked to a human heavychain constant region gene sequence. In certain embodiments, the heavychain constant region gene sequence encodes a sequence selected from aC_(H)1, a hinge, a C_(H)2, a C_(H)3, and a combination thereof. In someembodiments, the V_(H) sequence and the J_(H) sequence are derived froma human V_(H) gene segment and a human J_(H) gene segment. In certainembodiments, wherein the human V_(H) gene segment is selected from thegroup consisting of V_(H)1-2, V_(H)1-3, V_(H)1-8, V_(H)1-18, V_(H)1-24,V_(H)1-45, V_(H)1-46, V_(H)1-58, V_(H)1-69, V_(H)2-5, V_(H)2-26,V_(H)2-70, V_(H)3-7, V_(H)3-9, V_(H)3-11, V_(H)3-13, V_(H)3-15,V_(H)3-16, V_(H)3-20, V_(H)3-21, V_(H)3-23, V_(H)3-30, V_(H)3-30-3,V_(H) 3-30-5, V_(H)3-33, V_(H)3-35, V_(H)3-38, V_(H)3-43, V_(H)3-48,V_(H)3-49, V_(H)3-53, V_(H)3-64, V_(H)3-66, V_(H)3-72, V_(H)3-73,V_(H)3-74, V_(H)4-4, V_(H)4-28, V_(H)4-30-1, V_(H)4-30-2, V_(H)4-30-4,V_(H)4-31, V_(H)4-34, V_(H)4-39, V_(H)4-59, V_(H)4-61, V_(H)5-51,V_(H)6-1, V_(H)7-4-1, V_(H)7-81, and a polymorphic variant thereof. Inparticular embodiments, the human V_(H) gene segment is V_(H)3-23 or apolymorphic variant thereof. In some embodiments, the spacer encodes asequence derived from a human D gene segment. In particular embodiments,the human D gene segment is selected from the group consisting of D1-1,D1-7, D1-14, D1-20, D1-26, D2-2, D2-8, D2-15, D2-21, D3-3, D3-9, D3-10,D3-16, D3-22, D4-4, D4-11, D4-17, D4-23, D5-12, D5-5, D5-18, D5-24,D6-6, D6-13, D6-19, D6-25, D7-27, and a polymorphic variant thereof. Inparticular embodiments, the spacer encodes the sequence of D4-4 or apolymorphic variant thereof. In certain embodiments, the human J_(H)gene segment is selected from the group consisting of J_(H)1, J_(H)2,J_(H)3, J_(H)4, J_(H)S, J_(H)6, and a polymorphic variant thereof. Incertain embodiments, the human J_(H) segment is J_(H)4-4 or apolymorphic variant thereof. In some embodiments, the rearrangedimmunoglobulin heavy chain variable region nucleotide sequence encodesthe sequence of human V_(H)3-23/GY/J_(H)4-4 (SEQ ID NO: 137). In someembodiments, substantially all endogenous functional V_(H), D, and J_(H)gene segments are deleted from the immunoglobulin heavy chain variablelocus of the non-human animal or rendered non-functional. In someembodiments, the non-human animal comprises a modification that deletesor renders non-functional endogenous functional V_(H), D, and J_(H) genesegments; and the non-human animal comprises the rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence at anectopic locus of its genome. In some embodiments, a heavy chain variabledomain encoded by the rearranged human immunoglobulin heavy chainvariable region nucleotide sequence is not immunogenic to the non-humananimal. In some embodiments, the non-human animal comprises an Adam6agene, an Adam6b gene, or both.

Additional aspects provide an immunoglobulin heavy chain locus in agermline genome of a non-human animal, comprising a rearranged humanimmunoglobulin heavy chain variable region nucleotide sequencecomprising a heavy chain variable gene segment (V_(H)) that is operablylinked, via a spacer, to a heavy chain J gene segment (J_(H)), whereinthe spacer encodes at least one amino acid residue. In some embodiments,the rearranged human immunoglobulin heavy chain variable regionnucleotide sequence is operably linked to a non-human heavy chainconstant region gene sequence. In certain embodiments, the non-humanheavy chain constant region gene sequence is a mouse or a rat heavychain constant region gene sequence. In some embodiments, theimmunoglobulin locus comprises a plurality of copies of the rearrangedheavy chain variable region nucleotide sequence.

In additional aspects are provided methods of making a non-human animalcomprising a modified immunoglobulin heavy chain locus, the methodscomprising:

-   -   (a) modifying a genome of a non-human animal to delete or render        non-functional endogenous functional immunoglobulin heavy chain        V_(H), D, and J_(H) gene segments; and    -   (b) placing in the genome a rearranged human immunoglobulin        heavy chain variable region nucleotide sequence comprising a        heavy chain variable gene segment (V_(H)) that is operably        linked, via a spacer, to a heavy chain J gene segment (J_(H)),        wherein the spacer comprises at least one amino acid residue.        In some embodiments, the rearranged human immunoglobulin heavy        chain nucleotide sequence is operably linked to a non-human        immunoglobulin heavy chain constant region gene sequence. In        certain embodiments, the non-human immunoglobulin heavy chain        constant region gene sequence is a mouse or a rat heavy chain        constant region gene sequence. In some embodiments, the        rearranged human immunoglobulin heavy chain variable region        nucleotide sequence is placed at an endogenous immunoglobulin        heavy chain locus in the genome. In some embodiments, the        rearranged human immunoglobulin heavy chain variable region        nucleotide sequence is present at an ectopic locus in the        genome. In some embodiments, the modified heavy chain locus        comprises a plurality of copies of the rearranged human        immunoglobulin heavy chain variable region nucleotide sequence.        In some embodiments, the non-human animal is a rodent selected        from the group consisting of a mouse, a rat, or a hamster. In        some embodiments, the non-human animal comprises an Adam6a gene,        an Adam6b gene or both. In some embodiments, the modified        immunoglobulin heavy chain locus is present in a germline genome        of the non-human animal.

Additional aspects provide a modified non-human animal comprising in itsgenome:

-   -   (a) an immunoglobulin heavy chain locus that comprises a        rearranged human heavy chain variable region nucleic acid        sequence operably linked to a heavy chain constant region        nucleic acid sequence; and    -   (b) an immunoglobulin light chain locus comprising one or more        but less than the wild type number of human immunoglobulin light        chain V_(L) and J_(L) gene segments, operably linked to a light        chain constant region nucleic acid sequence.        In some embodiments, the non-human animal is a mammal. In        certain embodiments, the mammal is a rodent. In particular        embodiments, the rodent is selected from the group consisting of        a mouse, a rat, and a hamster. In some embodiments, the heavy        chain constant region nucleic acid sequence is a rodent constant        region sequence that encodes an immunoglobulin isotype selected        from IgM, IgD, IgG, IgE, IgA, and a combination thereof. In some        embodiments, the light chain constant region nucleic acid        sequence is a rodent κ or λ constant region nucleic acid        sequence. In some embodiments, the rearranged human heavy chain        variable region nucleic acid sequence is selected from a human        germline V_(H) segment, a human germline D segment, and a human        germline J_(H) segment. In some embodiments, the rearranged        human heavy chain variable region nucleic acid sequence is        operably linked to the constant region nucleic acid sequence at        an endogenous locus. In some embodiments, the one or more but        less than the wild type number of human immunoglobulin light        chain V_(L) and J_(L) gene segments are operably linked to the        light chain constant region nucleic acid sequence at an        endogenous locus. In some embodiments, the non-human animal        comprises no more than two human immunoglobulin unrearranged        V_(L) gene segments, and one, two, or three or more human        unrearranged J_(L) gene segments. In some embodiments, the        rearranged heavy chain variable region nucleic acid sequence is        derived from a human germline V_(H) gene segment selected from        the group consisting of V_(H)1-2, V_(H)1-3, V_(H)1-8, V_(H)1-18,        V_(H)1-24, V_(H)1-45, V_(H)1-46, V_(H)1-58, V_(H)1-69, V_(H)2-5,        V_(H)2-26, V_(H)2-70, V_(H)3-7, V_(H)3-9, V_(H)3-11, V_(H)3-13,        V_(H)3-15, V_(H)3-16, V_(H)3-20, V_(H)3-21, V_(H)3-23,        V_(H)3-30, V_(H)3-30-3, V_(H) 3-30-5, V_(H)3-33, V_(H)3-35,        V_(H)3-38, V_(H)3-43, V_(H)3-48, V_(H)3-49, V_(H)3-53,        V_(H)3-64, V_(H)3-66, V_(H)3-72, V_(H)3-73, V_(H)3-74, V_(H)4-4,        V_(H)4-28, V_(H)4-30-1, V_(H)4-30-2, V_(H)4-30-4, V_(H)4-31,        V_(H)4-34, V_(H)4-39, V_(H)4-59, V_(H)4-61, V_(H)5-51, V_(H)6-1,        V_(H)7-4-1, V_(H)7-81, and a polymorphic variant thereof. In        certain, the rearranged heavy chain variable region nucleic acid        sequence is derived from a human germline V_(H)3-23 gene        segment. In some embodiments, the rearranged heavy chain        variable region nucleic acid sequence encodes the sequence of        human V_(H)3-23/GY/J_(H)4-4 (SEQ ID NO: 137). In some        embodiments, the rearranged heavy chain variable region nucleic        acid sequence encodes the sequence of V_(H)3-23/X₁X₂/J, wherein        X₁ is any amino acid, and X₂ is any amino acid. In certain        embodiments, X₁ is Gly and X₂ is Tyr. In some embodiments, the        immunoglobulin heavy chain locus comprises a functional Adam6a        gene, Adam6b gene, or both. In certain embodiments, the Adam6a        gene, Adam6b gene, or both are endogenous Adam6 genes. In some        embodiments, the genetically modified non-human animal comprises        an Adam6a gene, Adam6b gene, or both at an ectopic locus of the        genome. In some embodiments, the human variable region V_(L) and        J_(L) gene segments are capable of rearranging and encoding a        human immunoglobulin light chain variable domain. In some        embodiments, the immunoglobulin light chain locus comprises two        human V_(L) gene segments, V_(κ)1-39 and V_(κ)3-20. In certain        embodiments, the gene segments are germline gene segments. In        certain embodiments, the non-human animal comprises J_(κ)1,        J_(κ)2, J_(κ)3, J_(κ)4, and J_(κ)5 gene segments. In some        embodiments, two or more, three or more, four or more, or five        or more human V_(L) gene segments and two or more human J_(L)        gene segments are present at an endogenous light chain locus. In        some embodiments, at least one of the human light chain V_(L) or        J_(L) gene segments encode one or more histidine codons that are        not encoded by a corresponding human germline light chain        variable gene segment. In some embodiments, at least one of the        V_(L) gene segments comprises an addition or substitution of at        least one non-histidine codon encoded by the corresponding human        germline V_(L) segment sequence with a histidine codon. In        certain embodiments, the added or substituted histidine codon is        present in CDR3. In some embodiments, the human V_(L) gene        segments are human V_(κ)1-39 and V_(κ)3-20 gene segments, and        each of the human V_(κ)1-39 and V_(κ)3-20 gene segments        comprises a substitution of at least one non-histidine codon        encoded by a corresponding human germline V_(L) gene segment        with the histidine codon. In certain embodiments, the        substitution is of three non-histidine codons of the human        V_(κ)1-39 gene segment, wherein the substitution is designed to        express histidines at positions 106, 108, and 111. In particular        embodiments, the substitution is of four non-histidine codons of        the human V_(κ)1-39 gene segment, and the substitution is        designed to express histidines at positions 105, 106, 108,        and 111. In particular embodiments, the substitution is of three        non-histidine codons of the human V_(κ)3-20 gene segment, and        the substitution is designed to express histidines at positions        105, 106, and 109. In particular embodiments, the substitution        is of four non-histidine codons of the human V_(κ)3-20 gene        segment, and the substitution is designed to express histidines        at positions 105, 106, 107, and 109. In some embodiments,        non-human animal of claim 107, wherein the non-human animal,        upon stimulation by an antigen of interest, expresses an        antigen-binding protein that specifically binds the antigen,        wherein the antigen-binding protein comprises an amino acid        sequence derived from the human V_(L) and J_(L) gene segments,        and wherein the antigen-binding protein comprises at least one        histidine residue at an amino acid position encoded by the human        V_(L) gene segment. In some embodiments, the non-human animal        expresses a population of antigen-binding proteins in response        to an antigen, wherein all antigen-binding proteins in the        population comprise: (a) immunoglobulin heavy chains comprising        human heavy chain variable domains derived from the rearranged        human variable region nucleic acid sequence; and (b)        immunoglobulin light chains comprising immunoglobulin light        chain variable domains derived from a rearrangement of the human        V_(L) gene segments and the J_(L) gene segments in the genome of        the non-human animal, and wherein at least one of the human        V_(L) gene segments encodes one or more histidine codons that        are not encoded by the corresponding human germline V_(L) gene        segment.

Additional aspects provide methods for making a non-human animalcomprising a modified immunoglobulin locus, comprising:

-   -   (a) modifying a genome of a non-human animal to delete or render        non-functional:        -   (i) endogenous functional immunoglobulin heavy chain V_(H),            D, and/or J_(H) gene segments, and        -   (ii) endogenous functional immunoglobulin light chain V_(L)            and J_(L) gene segments; and    -   (b) placing in the modified genome of the non-human animal:        -   (i) a rearranged human immunoglobulin heavy chain variable            region nucleic acid sequence in operable linkage to an            immunoglobulin heavy chain constant region nucleic acid            sequence; and        -   (ii) one or more but less than the wild type number of human            immunoglobulin light chain V_(L) and J_(L) gene segments in            operable linkage to an immunoglobulin light chain constant            region nucleic acid sequence.

In some embodiments, the non-human animal is a rodent. In certainembodiments, the rodent is a mouse, a rat, or a hamster. In someembodiments, the rearranged human immunoglobulin heavy chain variableregion nucleic acid sequence is operably linked to a mouse or rat heavychain constant region gene sequence selected from a CH1, a hinge, a CH2,a CH3, and a combination thereof. In some embodiments, the rearrangedhuman immunoglobulin heavy chain variable region nucleic acid sequenceand the human immunoglobulin light chain V_(L) and J_(L) gene segmentsare placed at or near a corresponding nucleotide sequence of the wildtype non-human animal. In some embodiments, the rearrangedimmunoglobulin human heavy chain variable region nucleic acid sequenceand the human immunoglobulin light chain V_(L) and J_(L) gene segmentsare placed at an ectopic locus in the genome. In some embodiments, thenon-human animal comprises an immunoglobulin heavy chain locuscomprising an endogenous Adam6a gene, Adam6b gene, or both. In someembodiments, the non-human animal comprises an Adam6a gene, Adam6b gene,or both at an ectopic locus of the genome. In some embodiments, therearranged human immunoglobulin heavy chain variable region nucleic acidsequence encodes the sequence of human V_(H)3-23/GY/J_(H)4-4 (SEQ ID NO:137). In some embodiments, the immunoglobulin light chain constantregion nucleic acid sequence is a rat or a mouse Cκ constant regionnucleic acid sequence. In some embodiments, the human immunoglobulinlight chain V_(L) and J_(L) gene segments are capable of rearranging andencoding a human immunoglobulin light chain variable domain. In someembodiments, the non-human animal comprises an immunoglobulin lightchain locus comprising two human V_(L) gene segments, V_(κ)1-39 andV_(κ)3-20. In certain embodiments, the non-human animal comprisesJ_(κ)1, J_(κ)2, J_(κ)3, J_(κ)4, and J_(κ)5 gene segments. In someembodiments, two or more, three or more, four or more, or five or morehuman V_(L) gene segments and two or more human J_(L) gene segments arepresent at an endogenous light chain locus. In some embodiments, atleast one of the human immunoglobulin light chain V_(L) or J_(L) genesegments encode one or more histidine codons that are not encoded by acorresponding human germline light chain variable gene segment. In someembodiments, at least one of the V_(L) gene segments comprises anaddition or a substitution of at least one non-histidine codon encodedby the corresponding human germline V_(L) segment sequence with ahistidine codon. In certain embodiments, the added or substitutedhistidine codon is present in CDR3. In certain embodiments, the humanV_(L) gene segments are human V_(κ)1-39 and V_(κ)3-20 gene segments, andeach of the human V_(κ)1-39 and V_(κ)3-20 gene segments comprises thesubstitution of at least one non-histidine codon encoded by acorresponding human germline V_(L) gene segment with the histidinecodon. In particular embodiments, the substitution is of threenon-histidine codons of the human V_(κ)1-39 gene segment, and whereinthe substitution is designed to express histidines at positions 106,108, and 111. In particular embodiments, the substitution is of fournon-histidine codons of the human V_(κ)1-39 gene segment, and whereinthe substitution is designed to express histidines at positions 105,106, 108, and 111. In particular embodiments, the substitution is ofthree non-histidine codons of the human V_(κ)3-20 gene segment, andwherein the substitution is designed to express histidines at positions105, 106, and 109. In particular embodiments, the substitution is offour non-histidine codons of the human V_(κ)3-20 gene segment, and thesubstitution is designed to express histidines at positions 105, 106,107, and 109.

Additional aspects provide methods for obtaining a nucleic acid sequencethat encodes an immunoglobulin light chain variable domain (V_(L))capable of binding an antigen independently from a heavy chain variabledomain, comprising:

-   -   (a) immunizing a non-human animal with an antigen of interest or        an immunogen thereof, wherein the non-human animal comprises in        its genome (i) a rearranged human immunoglobulin heavy chain        variable region nucleic acid sequence operably linked to a heavy        chain constant region nucleic acid sequence; and (ii) two or        more but less than the wild type number of human immunoglobulin        light chain variable region gene segments (V_(L) and J_(L))        operably linked to a light chain constant region nucleic acid        sequence;    -   (b) allowing the non-human animal to mount an immune response;    -   (c) isolating from the immunized non-human animal a cell        comprising a nucleic acid sequence that encodes a light chain        variable domain that can bind the antigen; and    -   (d) obtaining from the cell a nucleic acid sequence that encodes        the light chain variable domain (V_(L) domain) that can bind the        antigen.

In some embodiments, the isolating step (c) is carried out viafluorescence-activated cell sorting (FACS) or flow cytometry. In someembodiments, the cell comprising the nucleic acid sequence that encodesthe light chain variable domain that bind the antigen is a lymphocyte.In certain embodiments, the lymphocyte comprises natural killer cells, Tcells, or B cells. In some embodiments, the method further comprises astep of (c)′ fusing the lymphocyte with a cancer cell. In certainembodiments, the cancer cell is a myeloma cell. In some embodiments, thenucleic acid sequence of (d) is fused with a nucleic acid sequenceencoding an immunoglobulin constant region nucleic acid sequence. Insome embodiments, the light chain constant region nucleic acid sequenceis a human kappa sequence or a human lambda sequence. In someembodiments, the heavy chain constant region nucleic acid sequence is ahuman sequence selected from a CH1, a hinge, a CH2, a CH3, and acombination thereof. In some embodiments, the nucleic acid sequence of(d) comprises one or more histidine codon substitutions or insertionsthat are derived from the unrearranged V_(L) gene segment in the genomeof the animal.

Additional aspects provide methods for making an antigen-binding proteinthat comprises an immunoglobulin light chain variable domain that canbind an antigen independently from a heavy chain variable domain,comprising:

-   -   (a) immunizing a genetically modified non-human animal with a        first antigen that comprises a first epitope or immunogenic        portion thereof, wherein the non-human animal comprises in its        genome:        -   (i) a rearranged human heavy chain variable region nucleic            acid sequence operably linked to a heavy chain constant            region nucleic acid sequence; and        -   (ii) two or more but less than the wild type number of human            immunoglobulin light chain variable region gene segments            (V_(L) and J_(L)) operably linked to an immunoglobulin light            chain constant region nucleic acid sequence;    -   (b) allowing the non-human animal to mount an immune response to        the first epitope or immunogenic portion thereof;    -   (c) isolating from the non-human animal a cell comprising a        nucleic acid sequence that encodes a light chain variable domain        that specifically binds the first epitope or immunogenic portion        thereof;    -   (d) obtaining from the cell of (c) the nucleic acid sequence        that encodes the light chain variable domain that specifically        binds the first epitope or immunogenic portion thereof;    -   (e) employing the nucleic acid sequence of (d) in an expression        construct, fused to a human immunoglobulin constant region        nucleic acid sequence; and    -   (f) expressing the nucleic acid sequence of (d) in a production        cell line that expresses a human immunoglobulin heavy chain that        specifically binds a second antigen or epitope to form an        antigen-binding protein whose light chain is encoded by the        nucleic acid of (d) and that binds the first epitope or        immunogenic portion thereof independently from the heavy chain,        and whose heavy chain specifically binds the second antigen or        epitope.

In some embodiments, at least one of the human light chain V_(L) orJ_(L) gene segments encode one or more histidine codons that are notencoded by a corresponding human germline light chain variable genesegment. In some embodiments, the first epitope is derived from a cellsurface receptor. In particular embodiments, the cell surface receptoris an Fc receptor. In yet more particular embodiments, the Fc receptoris FcRn. In some embodiments, the second antigen or epitope is derivedfrom a soluble antigen. In some embodiments, the second antigen orepitope is derived from a cell surface receptor. In some embodiments,the first antigen is an Fc receptor, the second antigen is a solubleprotein, and the antigen-binding protein comprises one or more histidinesubstitutions and insertions derived from the V_(L) gene segment in thegenome of the non-human animal.

Additional aspects provide methods for obtaining a nucleic acid sequencethat encodes an immunoglobulin light chain variable domain (V_(L))capable of binding an antigen independently from a heavy chain variabledomain, comprising:

-   -   (a) immunizing a non-human animal with a first antigen that        comprises a first epitope or immunogenic portion thereof,        wherein the non-human animal comprises in its genome: (i) a        rearranged human immunoglobulin heavy chain variable region        nucleic acid sequence operably linked to a heavy chain constant        region nucleic acid sequence, and (ii) an unrearranged human        immunoglobulin light chain variable region nucleic acid sequence        operably linked to a light chain constant region nucleic acid        sequence;    -   (b) allowing the non-human animal to mount an immune response;    -   (c) isolating from the immunized non-human animal a cell        comprising a nucleic acid sequence that encodes a light chain        variable domain that can bind the antigen; and    -   (d) obtaining from the cell a nucleic acid sequence that encodes        the light chain variable domain (V_(L) domain) that can bind the        antigen.

In some embodiments, the isolating step (c) is carried out viafluorescence-activated cell sorting (FACS) or flow cytometry. In someembodiments, the cell comprising the nucleic acid sequence that encodesthe light chain variable domain that binds the antigen is a lymphocyte.In certain embodiments, the lymphocyte comprises natural killer cells, Tcells, or B cells. In some embodiments, the method further comprises:(c)′ fusing the lymphocyte with a cancer cell. In certain embodiments,the cancer cell is a myeloma cell. In some embodiments, the nucleic acidsequence of (d) is fused with a nucleic acid sequence encoding animmunoglobulin constant region nucleic acid sequence. In someembodiments, the light chain constant region nucleic acid sequence is ahuman kappa sequence or a human lambda sequence. In some embodiments,the heavy chain constant region nucleic acid sequence is a humansequence selected from a CH1, a hinge, a CH2, a CH3, and a combinationthereof. In some embodiments, the nucleic acid sequence of (d) comprisesone or more histidine codon substitutions or insertions that are derivedfrom the unrearranged V_(L) gene segment in the genome of the animal.

Additional aspect provide methods for obtaining a nucleic acid sequencethat encodes an immunoglobulin light chain variable domain (V_(L))capable of binding an antigen independently from a heavy chain variabledomain, comprising:

-   -   (a) immunizing a non-human animal with a first antigen that        comprises a first epitope or immunogenic portion thereof,        wherein the non-human animal comprises in its genome: (i) a        rearranged human immunoglobulin heavy chain variable region        nucleic acid sequence operably linked to a light chain constant        region nucleic acid sequence, and (ii) human immunoglobulin        light chain variable region gene segments (V_(L) and J_(L))        operably linked to a heavy chain constant region nucleic acid        sequence;    -   (b) allowing the non-human animal to mount an immune response;    -   (c) isolating from the immunized non-human animal a cell        comprising a nucleic acid sequence that encodes a light chain        variable domain that can bind the antigen; and    -   (d) obtaining from the cell a nucleic acid sequence that encodes        the light chain variable domain (V_(L) domain) that can bind the        antigen.

In some embodiments, the isolating step (c) is carried out viafluorescence-activated cell sorting (FACS) or flow cytometry. In someembodiments, the cell comprising the nucleic acid sequence that encodesthe light chain variable domain that binds the antigen is a lymphocyte.In certain embodiments, the lymphocyte comprises natural killer cells, Tcells, or B cells. In some embodiments, the method further comprises:(c)′ fusing the lymphocyte with a cancer cell. In certain embodiments,the cancer cell is a myeloma cell. In some embodiments, the nucleic acidsequence of (d) is fused with a nucleic acid sequence encoding animmunoglobulin constant region nucleic acid sequence. In someembodiments, the light chain constant region nucleic acid sequence is ahuman kappa sequence or a human lambda sequence. In some embodiments,the heavy chain constant region nucleic acid sequence is a humansequence selected from a CH1, a hinge, a CH2, a CH3, and a combinationthereof. In some embodiments, the nucleic acid sequence of (d) comprisesone or more histidine codon substitutions or insertions that are derivedfrom the unrearranged V_(L) gene segment in the genome of the animal.

Additional aspect provided methods for making an antigen-binding proteinthat comprises an immunoglobulin light chain variable domain that canbind an antigen independently from a heavy chain variable domain,comprising:

-   -   (a) immunizing a genetically modified non-human animal with a        first antigen that comprises a first epitope or immunogenic        portion thereof, wherein the non-human animal comprises in its        genome:        -   (i) a rearranged human heavy chain variable region nucleic            acid sequence operably linked to a heavy chain constant            region nucleic acid sequence; and        -   (ii) unrearranged human immunoglobulin light chain variable            region gene segments (V_(L) and J_(L)) operably linked to a            light chain constant region nucleic acid sequence;    -   (b) allowing the non-human animal to mount an immune response to        the first epitope or immunogenic portion thereof;    -   (c) isolating from the non-human animal a cell comprising a        nucleic acid sequence that encodes a light chain variable domain        that specifically binds the first epitope or immunogenic portion        thereof;    -   (d) obtaining from the cell of (c) the nucleic acid sequence        that encodes the light chain variable domain that specifically        binds the first epitope or immunogenic portion thereof;    -   (e) employing the nucleic acid sequence of (d) in an expression        construct, fused to a human immunoglobulin constant region        nucleic acid sequence; and    -   (f) expressing the nucleic acid sequence of (d) in a production        cell line that expresses a human immunoglobulin heavy chain that        specifically binds a second antigen or epitope to form an        antigen-binding protein whose light chain is encoded by the        nucleic acid of (d) and that binds the first epitope or        immunogenic portion thereof independently from the heavy chain,        and whose heavy chain specifically binds the second antigen or        epitope.

In some embodiments, at least one of the human light chain V_(L) orJ_(L) gene segments encode one or more histidine codons that are notencoded by a corresponding human germline light chain variable genesegment. In some embodiments, the first epitope is derived from a cellsurface receptor. In certain embodiments, the cell surface receptor isan Fc receptor. In particular embodiments, the Fc receptor is FcRn. Insome embodiments, the second antigen or epitope is derived from asoluble antigen. In some embodiments, the second antigen or epitope isderived from a cell surface receptor. In some embodiments, the firstantigen is an Fc receptor, the second antigen is a soluble protein, andthe antigen-binding protein comprises one or more histidinesubstitutions and insertions derived from the V_(L) gene segment in thegenome of the non-human animal.

Additional aspects provided methods for making an antigen-bindingprotein that comprises an immunoglobulin light chain variable domainthat can bind an antigen independently from a heavy chain variabledomain, comprising:

-   -   (a) immunizing a genetically modified non-human animal with a        first antigen that comprises a first epitope or immunogenic        portion thereof, wherein the non-human animal comprises in its        genome:        -   (i) a rearranged human heavy chain variable region nucleic            acid sequence operably linked to a light chain constant            region nucleic acid sequence; and        -   (ii) unrearranged human immunoglobulin light chain variable            region gene segments (V_(L) and J_(L)) operably linked to a            heavy chain constant region nucleic acid sequence;    -   (b) allowing the non-human animal to mount an immune response to        the first epitope or immunogenic portion thereof;    -   (c) isolating from the non-human animal a cell comprising a        nucleic acid sequence that encodes a light chain variable domain        that specifically binds the first epitope or immunogenic portion        thereof;    -   (d) obtaining from the cell of (c) the nucleic acid sequence        that encodes the light chain variable domain that specifically        binds the first epitope or immunogenic portion thereof;    -   (e) employing the nucleic acid sequence of (d) in an expression        construct, fused to a human immunoglobulin constant region        nucleic acid sequence; and    -   (f) expressing the nucleic acid sequence of (d) in a production        cell line that expresses a human immunoglobulin heavy chain that        specifically binds a second antigen or epitope to form an        antigen-binding protein whose light chain is encoded by the        nucleic acid of (d) and that binds the first epitope or        immunogenic portion thereof independently from the heavy chain,        and whose heavy chain specifically binds the second antigen or        epitope.        In some embodiments, at least one of the human light chain V_(L)        or J_(L) gene segments encode one or more histidine codons that        are not encoded by a corresponding human germline light chain        variable gene segment. In some embodiments, the first epitope is        derived from a cell surface receptor. In certain embodiments,        the cell surface receptor is an Fc receptor. In particular        embodiments, the Fc receptor is FcRn. In some embodiments, the        second antigen or epitope is derived from a soluble antigen. In        some embodiments, the second antigen or epitope is derived from        a cell surface receptor. In some embodiments, the first antigen        is an Fc receptor, the second antigen is a soluble protein, and        the antigen-binding protein comprises one or more histidine        substitutions and insertions derived from the VL gene segment in        the genome of the non-human animal.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 illustrates schemes for constructing a rearranged heavy chainvariable domain mini-locus (“UHC mini-locus”) comprising a rearrangedhuman immunoglobulin variable region nucleotide sequence(V_(H)3-23/D/J_(H)4; SEQ ID NO: 136) and an intron of J_(H)4 (SEQ ID NO:140), which are operably linked to a human V_(H)3-23 promoter (SEQ IDNO: 139). The UHC mini-locus was flanked 5′ and 3′ by mouse homologyarms. In Step 1 (I-CeuI/SpeI Ligation (Amp+Spec)), a spectinomycinselection cassette was introduced into the upstream of the promoterbetween the I-CeuI and SpeI sites to generate pJSh0038 (UHC mini-locus).

FIG. 2 illustrates schemes for (A) targeting a hygromycin selectioncassette (EM7-HYG) into the 5′ end of the MAID 1115 BAC clone (2. BHR(Hyg+Kan)); and (B) targeting the UHC mini-locus (pJSh0038) into theupstream of the IgM locus in the VI432 BAC clone (3. BHR (Spec+Hyg)).

FIG. 3 illustrates schemes for (A) targeting the pDBa0049 constructcomprising a chloramphenicol cassette into the 3′ end of the VI421clone, which comprises, from 5′ to 3′, an Adam6a gene (present in a 3′to 5′ direction); a neomycin cassette (present in a 3′ to 5′ direction)flanked by FRT sites; an Adam6b gene (present in a 3′ to 5′ direction);Intergenic Control Region 1 (IGCR1; a key V(D)J recombination regulatoryregion); and a spectinomycin cassette (present in a 5′ to 3′ direction)(4. BHR (Cm+Kan)); and (B) targeting the genomic locus of the VI444 BACclone containing the Adam6a and 6b genes into the upstream of theuniversal heavy chain (UHC) genomic locus of the VI443 BAC clone betweenthe I-CeuI and the AscI sites via restriction digestion and ligation (5.I-CeuI/AscI ligation (Hyg+Kan)).

FIG. 4 illustrates schemes for (A) targeting the final construct(MAID6031 BAC DNA) into ES cells isolated from the 1661 heterozygousmouse; and shows (B) the genomic location of the probes and primers usedin the screening assays.

FIG. 5 shows a list of antibodies in the ASAP database of RegeneronPharmaceuticals that contain CDR3 sequences similar to the UHC CDR3sequence (AKDYSNYYFDY; SEQ ID NO: 143).

FIG. 6 illustrates the genomic organization of the 6031 bacterialartificial chromosome (BAC) DNA and 6031 heterozygous ES cells, and thegenomic location of the primers and probes used in the screening assays.

FIG. 7 shows a list of primers and probes used to confirm a loss ofallele (LOA), a gain of allele (GOA), or a parental allele (parental) inthe screening assays.

FIG. 8 shows sequences of primers and probes used in the screeningassays.

FIG. 9 illustrates the genomic structure of the immunoglobulin heavychain locus of genetically modified FO mice, which contains one copy ofthe targeted allele (including the Adam6a/6b genes and the rearrangedhuman immunoglobulin heavy chain variable region nucleotide sequence(hV_(H)3-23(D)J_(H)4). (A) MAID 6031 het: a heterozygous FO mousecomprising a genetically modified immunoglobulin heavy chain locus witha selection cassette; (B) MAID 6032 het: a heterozygous FO mousecomprising a genetically modified immunoglobulin heavy chain locuswithout a selection cassette.

FIG. 10 shows the result of fluorescence-activated cell sorting (FACS)analysis of the bone marrow cells isolated from a wild type or 6032heterozygous mouse. Upper Panel: Bone marrow cells isolated from a wildtype or an FO 6032 heterozygous mouse were gated on singlets and sortedbased on CD19 expression (a B cell marker) and CD3 expression (a T cellmarker). Lower Panel: CD19+-gated B cells were sorted based on thepresence of IgM^(b) antibodies (antibodies produced from a wild typeallele; B6 allele) or IgM^(a) antibodies (antibodies produced from thegenetically modified allele (129 allele) encoding a rearranged heavychain variable domain (hV_(H)3-23(D)J_(H)4).

FIG. 11 shows the result of FACS analysis of the spleen cells isolatedfrom a wild type or 6032 heterozygous mouse. Upper Panel: Spleen cellsisolated from a wild type or FO 6032 heterozygous mouse were gated onsinglets and sorted based on CD19 expression (a B cell marker) and CD3expression (a T cell marker). Lower Panel: CD19+-gated B cells weresorted based on the presence of IgM^(b) antibodies (antibodies producedfrom a wild type allele; B6 allele) or IgM^(a) antibodies (antibodiesproduced from the genetically modified allele (129 allele) encoding arearranged heavy chain variable domain (hV_(H)3-23(D)J_(H)4).

FIG. 12 shows the result of FACS analysis of the blood cells isolatedfrom a wild type or 6032 heterozygous mouse. Upper Panel: Blood cellsisolated from a wild type or FO 6032 heterozygous mouse were gated onsinglets and sorted based on CD19 expression (a B cell marker) and CD3expression (a T cell marker). Lower Panel: CD19+-gated B cells weresorted based on the presence of IgM^(b) antibodies (antibodies producedfrom a wild type allele; B6 allele) or IgM^(a) antibodies (antibodiesproduced from the genetically modified allele (129 allele) encoding arearranged heavy chain variable domain (hV_(H)3-23(D)J_(H)4).

FIG. 13A shows the results of FACS analysis for the total number ofCD19+ B cells immature B cells (CD19+IgD^(int)IgMhi) and mature B cells(CD19+IgM^(lo)IgD^(hi)) in harvested spleens from wild type mice (WT)and mice homozygous (6032HO) for a rearranged human immunoglobulinvariable region nucleotide sequence (V_(H)3-23/D/J_(H)4). Upper Panel:Spleen cells isolated from a wild type or F2 6032 homozygous mouse weregated on singlets and sorted based on CD19 expression (a B cell marker)and CD3 expression (a T cell marker). The bottom panel showsrepresentative contour plots of splenocytes gated on CD19+ and stainedfor immunoglobulin D (IgD) and immunoglobulin M (IgM) from a wild typemouse (WT) and a mouse homozygous for a rearranged heavy chain humanimmunoglobulin variable region nucleotide sequence (V_(H)3-23/D/J_(H)4).Percentage of cells within each gated region is shown.

FIG. 13B shows the total number of B cells (CD19+), mature B cells(CD19+IgD^(hi) IgM^(lo)) and immature B cells (CD19+IgD^(int)IgM^(hi))in harvested spleens from wild type (WT) and mice homozygous for arearranged human immunoglobulin heavy chain variable region nucleotidesequence (V_(H)3-23/D/J_(H)4).

FIG. 13C shows representative contour plots of Igλ+ and Igκ+ splenocytesgated on CD19+ from a wild type mouse (WT) and a mouse (6032HO)homozygous for a rearranged human immunoglobulin heavy chain variableregion nucleotide sequence (V_(H)3-23/D/J_(H)4).

FIG. 13D shows the total number of B cells (CD19+), Igκ+ B cells(CD19+Igkappa+) and Igλ+ B cells (CD19+Iglambda+) in harvested spleensfrom wild type (WT) and mice homozygous for a rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence(V_(H)3-23/D/J_(H)4).

FIG. 13E shows the peripheral B cell development in a wild type mouseand mice homozygous for a rearranged human immunoglobulin heavy chainvariable region nucleotide sequence (V_(H)3-23/D/J_(H)4). The firstcolumn (left) of contour plots show CD93+ and B220+ splenocytes gated onCD19+ indicating immature and mature B cells. The second column (middle)of contour plot shows IgM+ and CD23+ expression in immature B cellsindicating T1 (IgD-IgM+CD21^(lo)CD23−), T2(IgD^(hi)IgM^(hi)CD21^(mid)CD23+) and T3 B cell populations. The thirdcolumn (right) of contour plots shows CD21+ (CD35+) and IgM+ expressionof mature B cells indicating a smaller first population that give riseto marginal zone B cells and a second population that gives rise tofollicular (FO) B cells. Percentage of cells within each gated region isshown.

FIG. 14A shows representative contour plots of bone marrow stained for Band T cells (CD19+ and CD3+, respectively) from a wild type mouse (WT)and a mouse homozygous for a rearranged human immunoglobulin heavy chainvariable region nucleotide sequence (V_(H)3-23/D/J_(H)4).

FIG. 14B shows the absolute number of cells (left), the total number ofcells (middle) and the total number of B (CD19+) cells (right) in bonemarrow isolated from the femurs of wild type mice (WT) and micehomozygous for rearranged human immunoglobulin heavy chain variableregion nucleotide sequence (V_(H)3-23/D/J_(H)4).

FIG. 14C shows representative contour plots of bone marrow gated onsinglets stained for immunoglobulin M (IgM) and B220 from a wild typemouse (WT) and a mouse homozygous for a rearranged human immunoglobulinheavy chain variable region nucleotide sequence (V_(H)3-23/D/J_(H)4).Immature, mature and pro/pre B cells are noted on each of the contourplots.

FIG. 14D shows the total number and mature B (B220^(hi)IgM+) andimmature B (B220^(int)IgM+) cells in bone marrow isolated from thefemurs of wild type mice (WT) and mice homozygous for a rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence(V_(H)3-23/D/J_(H)4).

FIG. 14E shows representative contour plots of bone marrow gated onsinglets stained for immunoglobulin M (IgM) and B220 from a wild typemouse (WT) and mice homozygous for a rearranged human immunoglobulinheavy chain variable region nucleotide sequence (V_(H)3-23/D/J_(H)4).Immature, mature and pro/pre B cells are noted on each of the contourplots.

FIG. 14F shows representative contour plots of bone marrow gated onimmature (B220^(int)IgM+) and mature (B220^(hi)IgM+) B cells stained forIgκ and Igλ expression isolated from the femurs of a wild type mouse(WT) and mice homozygous for a rearranged human immunoglobulin heavychain variable region nucleotide sequence (V_(H)3-23/D/J_(H)4).

FIG. 15 shows the levels of antigen-specific mIgGs in the mouse sera(Wild type or 6031 HET F0 and F1) at Day 15 and Day 24 following footpadimmunization.

FIG. 16 shows codon-optimized nucleotide sequence and deduced amino acidsequence of hV_(H)3-23(D4-4_Reading Frame 3)J_(H)6 (SEQ ID NO: 145).

FIG. 17 shows codon-optimized nucleotide sequence and deduced amino acidsequence of hV_(H)3-23(D4-4_Reading Frame 2)J_(H)6 (SEQ ID NO: 146).

FIG. 18 shows codon-optimized nucleotide sequence and deduced amino acidsequence of hV_(H)3-23(D4-4_Reading Frame 3)J_(H)4 (SEQ ID NO: 147).

FIG. 19 shows codon-optimized nucleotide sequence and deduced amino acidsequence of hV_(H)3-23(D4-4_Reading Frame 2)J_(H)4 (SEQ ID NO: 148).

FIG. 20 shows examples of two genetically modified dual light chain(DLC) loci. The locus on the top (DLC-5J) contains an engineered humanDNA fragment containing two human Vκ gene segments and five human Jκgene segments. The locus on the bottom (DLC-1J) contains an engineeredhuman DNA fragment containing two human Vκ gene segments and one humanJκ gene segment. Each locus is capable of rearranging to form a human Vκregion operably linked to an endogenous light chain constant region(e.g., a Cκ). Immunoglobulin promoters (P, open arrow above locus),leader exons (L, short open arrows), and the two human Vκ gene segments(long open arrows), all flanked upstream (5′) by a neomycin cassettecontaining Frt recombination sites are shown. Recombination signalsequences engineered with each of the human gene segments (Vκ and Jκ)are indicated by open ovals juxtaposed with each gene segment. In mostembodiments, unless indicated otherwise, filled shapes and solid linesrepresent mouse sequences, and open shapes and double lines representhuman sequences. The diagrams are not presented to scale.

FIGS. 21A-21C show a general strategy for construction of a targetingvector for the engineering of an immunoglobulin kappa locus comprisingtwo human Vκ segments (hVκ1-39 and hVκ3-20) and one human Jκ segment(Jκ5), as well as mouse enhancers and IgκC arm. FIG. 21D showsintroduction of this targeting vector into ES cells and generation ofheterozygous mice with the same; while FIG. 21E shows deletion of theselection cassette in ES cells using FLP enzyme. In most embodiments,unless indicated otherwise, filled shapes and solid lines representmouse sequences, and open shapes and double lines represent humansequences. The diagrams are not presented to scale.

FIGS. 22A-22D show the nucleotide sequence (SEQ ID NO:82) of theengineered portion of immunoglobulin κ locus comprising two human Vκsegments (hVκ1-39 and hVκ3-20) and one human Jκ segment; the nucleotidesequence spans the engineered human sequence and comprising 100 basepairs of endogenous mouse sequence at both the 5′ and the 3′ end. Bottomof FIG. 22D explains different fonts used to depict various sequences.

FIGS. 23A-23B show a general strategy for construction of a targetingvector for the engineering of an immunoglobulin kappa locus comprisingtwo human Vκ segments (hVκ1-39 and hVκ3-20) and five human Jκ segments,as well as mouse enhancers and IgκC arm. FIG. 23C shows introduction ofthis targeting vector into ES cells and generation of heterozygous micewith the same; while FIG. 23D shows deletion of the selection cassettein ES cells using FLP enzyme. In most embodiments, unless indicatedotherwise, filled shapes and solid lines represent mouse sequences, andopen shapes and double lines represent human sequences. The diagrams arenot presented to scale.

FIGS. 24A-24D show the nucleotide sequence (SEQ ID NO:83) of theengineered immunoglobulin κ locus comprising two human Vκ segments(hVκ1-39 and hVκ3-20) and five human Jκ segments; the nucleotidesequence spans the engineered sequence and 100 base pairs of endogenousmouse sequence at both the 5′ and the 3′ end. Bottom of FIG. 24Dexplains different fonts used to depict various sequences.

FIG. 25A, in the top panel, shows representative contour plots of bonemarrow stained for B and T cells (CD19+ and CD3+, respectively) from awild type mouse (WT) and a mouse homozygous for two human Vκ and fivehuman Jκ gene segments (DLC-5J). The bottom panel shows representativecontour plots of bone marrow gated on CD19+ and stained for ckit+ andCD43+ from a wild type mouse (WT) and a mouse homozygous for two humanVκ and five human Jκ gene segments (DLC-5J). Pro and Pre B cells arenoted on the contour plots of the bottom panel.

FIG. 25B shows the number of Pro (CD19+CD43+ckit+) and Pre(CD19+CD43−ckit−) B cells in bone marrow harvested from the femurs ofwild type mice (WT) and mice homozygous for two human Vκ and five humanJκ gene segments (DLC-5J).

FIG. 26A shows representative contour plots of bone marrow gated onsinglets stained for immunoglobulin M (IgM) and B220 from a wild typemouse (WT) and a mouse homozygous for two human Vκ and five human Jκgene segments (DLC-5J). Immature, mature and pro/pre B cells are notedon each of the contour plots.

FIG. 26B shows the total number of B (CD19+), immature B(B220^(int)IgM+) and mature B (B220^(hi)IgM+) cells in bone marrowisolated from the femurs of wild type mice (WT) and mice homozygous fortwo human Vκ and five human Jκ gene segments (DLC-5J).

FIG. 27A shows representative contour plots of bone marrow gated onsinglets stained for immunoglobulin M (IgM) and B220 from a wild typemouse (WT) and a mouse homozygous for two human Vκ and five human Jκgene segments (DLC-5J). Immature, mature and pro/pre B cells are notedon each of the contour plots.

FIG. 27B shows representative contour plots of bone marrow gated onimmature (B220^(int)IgM+) and mature (B220^(hi)IgM+) B cells stained forIgκ and Igλ expression isolated from the femurs of a wild type mouse(WT) and a mouse homozygous for two human Vκ and five human Jκ genesegments (DLC-5J).

FIG. 28A, in the top panel, shows representative contour plots ofsplenocytes gated on singlets and stained for B and T cells (CD19+ andCD3+, respectively) from a wild type mouse (WT) and a mouse homozygousfor two human Vκ and five human Jκ gene segments (DLC-5J). The bottompanel shows representative contour plots of splenocytes gated on CD19+and stained for immunoglobulin D (IgD) and immunoglobulin M (IgM) from awild type mouse (WT) and a mouse homozygous for two human Vκ and fivehuman Jκ gene segments (DLC-5J). Mature (54 for WT, 56.9 for DLC-5J) andtransitional (23.6 for WT, 25.6 for DLC-5J) B cells are noted on each ofthe contour plots.

FIG. 28B shows the total number of CD19+ B cells, transitional B cells(CD19+IgM^(hi)IgD^(lo)) and mature B cells (CD19+IgM^(lo)IgD^(hi)) inharvested spleens from wild type mice (WT) and mice homozygous for twohuman Vκ and five human Jκ gene segments (DLC-5J).

FIG. 29A shows representative contour plots of Igλ+ and Igκ+ splenocytesgated on CD19+ from a wild type mouse (WT) and a mouse homozygous fortwo human Vκ and five human Jκ gene segments (DLC-5J).

FIG. 29B shows the total number of B cells (CD19+), Igκ+ B cells(CD19+Igκ+) and Igλ+ B cells (CD19+Igλ+) in harvested spleens from wildtype (WT) and mice homozygous for two human Vκ and five human Jκ genesegments (DLC-5J).

FIG. 30A shows the peripheral B cell development in mice homozygous fortwo human Vκ and five human Jκ gene segments. The first (far left)contour plot shows CD93+ and B220+ splenocytes gated on CD19+ indicatingimmature (39.6) and mature (57.8) B cells. The second (top middle)contour plot shows IgM+ and CD23+ expression in immature B cellsindicating T1 (33.7; IgD-IgM+CD21^(lo)CD23−), T2 (21.2;IgD^(hi)IgM^(hi)CD21^(mid)CD23+) and T3 (29.1) B cell populations. Thethird (bottom middle) contour plot shows CD21+ (CD35+) and IgM+expression of mature B cells indicating a small population (14.8) whichgive rise to marginal zone B cells and a second population (70.5) whichgives rise to follicular (FO) B cells. The fourth (top right) contourplot shows B220+ and CD23+ expression in mature B cells indicatingmarginal zone (90.5; MZ) and marginal zone precursor (7.3;IgM^(hi)IgD^(hi)CD21^(hi)CD23+) B cell populations. The fifth (bottomright) contour plot shows IgD+ and IgM+ expression in mature B cellsindicating FO-I (79.0; IgD^(hi)IgM^(lo)CD21^(mid)CD23+) and FO-II (15.1;IgD^(hi)IgM^(hi)CD21^(mid)CD23+) B cell populations. Percentage of cellswithin each gated region is shown.

FIG. 30B shows the peripheral B cell development in wild type mice. Thefirst (far left) contour plot shows CD93+ and B220+ splenocytes gated onCD19+ indicating immature (31.1) and mature (64.4) B cells. The second(top middle) contour plot shows IgM+ and CD23+ expression in immature Bcells indicating T1 (28.5; IgD-IgM+CD21^(lo)CD23−), T2 (28.7;IgD^(hi)IgM^(hi)CD21^(mid)CD23+) and T3 (30.7) B cell populations. Thethird (bottom middle) contour plot shows CD21+ (CD35+) and IgM+expression of mature B cells indicating a small population (7.69) whichgive rise to marginal zone B cells and a second population (78.5) whichgives rise to follicular (FO) B cells. The fourth (top right) contourplot shows B220+ and CD23+ expression in mature B cells indicatingmarginal zone (79.9; MZ) and marginal zone precursor (19.4;IgM^(hi)IgD^(hi)CD21^(hi)CD23+) B cell populations. The fifth (bottomright) contour plot shows IgD+ and IgM+ expression in mature B cellsindicating FO-I (83.6; IgD^(hi)IgM^(lo)CD21^(mid)CD23+) and FO-II (13.1;IgD^(hi)IgM^(hi)CD21^(mid)CD23+) B cell populations. Percentage of cellswithin each gated region is shown.

FIG. 31 shows the total number of transitional, marginal zone andfollicular B cell populations in harvested spleens of wild-type (WT) andmice homozygous for two human Vκ and five human Jκ gene segments(DLC-5J).

FIG. 32 shows the relative mRNA expression in bone marrow (y-axis) ofVκ3-20-derived and Vκ1-39-derived light chains in a quantitative PCRassay using probes specific for Vκ3-20 or Vκ1-39 gene segments in micehomozygous for a replacement of the endogenous Vκ and Jκ gene segmentswith human Vκ and Jκ gene segments (HK) (human light chain of aVELOCIMMUNE® mouse), wild type mice (WT), mice homozygous for two humanVκ gene segments and five human Jκ gene segments (DLC-5J) and micehomozygous for two human Vκ gene segments and one human Jκ gene segment(DLC-1J). Signals are normalized to expression of mouse Cκ. ND: notdetected.

FIG. 33 shows the relative mRNA expression in whole spleens (y-axis) ofVκ3-20-derived and Vκ1-39-derived light chains in a quantitative PCRassay using probes specific for Vκ3-20 or Vκ1-39 gene segments in micehomozygous for a replacement of the endogenous Vκ and Jκ gene segmentswith human Vκ and Jκ gene segments (HK) (human light chain of aVELOCIMMUNE® mouse), wild type mice (WT), mice homozygous for two humanVκ gene segments and five human Jκ gene segments (DLC-5J) and micehomozygous for two human Vκ gene segments and one human Jκ gene segment(DLC-1J). Signals are normalized to expression of mouse Cκ. ND: notdetected.

FIG. 34 shows the sequence and properties (% GC content, N, % mismatch,Tm) of selected mutagenesis primers used to engineer four histidineresidues into CDR3's of human Vκ1-39 and Vκ3-20 light chain sequence.SEQ ID NOs for these primers used in the Sequence Listing are includedin the Table below. F=forward primer, R=reverse primer.

FIG. 35A shows introduction of a targeting vector comprising two humanVκ light chain segments each substituted with four histidine residuesand five human Jκ into ES cells and generation of heterozygous mice withthe same; while FIG. 35B shows deletion of the selection cassette in EScells using FLPo enzyme. In most embodiments, unless indicatedotherwise, filled shapes and solid lines represent mouse sequences, andopen shapes and double lines represent human sequences. The diagrams arenot presented to scale.

FIG. 36 shows the sequence and properties (% GC content, N, % mismatch,Tm) of selected mutagenesis primers used to engineer three histidineresidues into CDR3's of human Vκ1-39 and Vκ3-20 light chain sequence.SEQ ID NOs for these primers used in the Sequence Listing are includedin the Table below. F=forward primer, R=reverse primer.

FIG. 37A shows introduction of a targeting vector comprising two humanVκ light chain segments each substituted with three histidine residuesand five human Jκ into ES cells and generation of heterozygous mice withthe same; while FIG. 37B shows deletion of the selection cassette in EScells using FLPo enzyme. In most embodiments, unless indicatedotherwise, filled shapes and solid lines represent mouse sequences, andopen shapes and double lines represent human sequences. The diagrams arenot presented to scale.

FIG. 38A shows alignment of amino acid sequence encoded by humangermline Vκ3-20 sequence (bottom sequence) with exemplary amino acidtranslation of IgM light kappa chain variable sequence expressed in amouse comprising two V kappa segments (Vκ3-20 and Vκ1-39), eachsubstituted with 3 histidine residues in CDR3 sequence (top sequence);the alignment shows IgM kappa chain variable sequence expressed in amouse that retained all three histidine substitutions introduced intothe germline sequence. FIG. 38B shows alignment of amino acid sequenceencoded by human germline Vκ1-39 sequence (bottom sequence in eachalignment) with exemplary amino acid translation of IgM light kappachain variable sequence expressed in a mouse comprising two V kappasegments (Vκ3-20 and Vκ1-39), each substituted with 3 histidine residuesin CDR3 sequence (top sequence in each alignment); top alignment showsIgM kappa chain variable sequence expressed in a mouse that retained allthree histidine modifications introduced into the germline sequence, thebottom alignment shows IgM kappa chain variable sequence expressed in amouse that retained two out of three histidine modifications introducedinto the germline sequence. In some embodiments, histidine introducedinto the last position of the Vκ may be lost during V-J rearrangement.

FIG. 39 illustrates the genomic structure of genetically modified F2mice comprising rearranged heavy chain variable region nucleic acidsequence in the heavy chain loci (MAID6032; “UHC mouse”) and furthercomprising genetically engineered light chain loci containing two humanVκ gene segments (e.g., a human Vκ1-39 and human Vκ3-20 gene segment)and five human Jκ gene segments (hJκ1-5; DLC-5J) (MAID 1912HO).

FIG. 40A, in the top panel, shows representative contour plots ofsplenocytes gated on singlets and stained for B and T cells (CD19⁺ andCD3⁺, respectively) from genetically modified control mice (VI3; 1293HO1460HO) and mice homozygous for a rearranged heavy chain variable regionnucleic acid sequence in the heavy chain loci and two human Vκ and fivehuman Jκ gene segments in the light chain loci (MAID 1912HO 6032 HO; DLCx UHC). The bottom panel shows representative contour plots ofsplenocytes gated on CD19⁺ and stained for immunoglobulin D (IgD) andimmunoglobulin M (IgM) from genetically modified control mice (VI3;1293HO 1460HO) and mice homozygous for a rearranged heavy chain variableregion nucleic acid sequence in the heavy chain loci and two human Vκand five human Jκ gene segments in the light chain loci (MAID 1912HO6032 HO; DLC x UHC). Mature and immature B cells are noted on each ofthe contour plots.

FIG. 40B shows the total number of CD19⁺ B cells, mature B cells(CD19⁺IgM^(lo)IgD^(hi)) and immature B cells (CD19⁺IgM^(hi)IgD^(int)) inharvested spleens from genetically modified control mice (VI3; 1293HO1460HO) and mice homozygous for a rearranged heavy chain variable regionnucleic acid sequence in the heavy chain loci and two human Vκ and fivehuman Jκ gene segments in the light chain loci (MAID 1912HO 6032 HO; DLCx UHC).

FIG. 41A shows representative contour plots of Igλ⁺ and Igκ⁺ splenocytesgated on CD19⁺ from genetically modified control mice (VI3; 1293HO1460HO) and mice homozygous for a rearranged heavy chain variable regionnucleic acid sequence in the heavy chain loci and two human Vκ and fivehuman Jκ gene segments in the light chain loci (MAID 1912HO 6032 HO; DLCx UHC).

FIG. 41B shows the total number of B cells (CD19⁺), Igκ⁺ B cells(CD19⁺Igκ⁺) and Igλ⁺ B cells (CD19⁺Igλ⁺) in harvested spleens fromgenetically modified control mice (VI3; 1293HO 1460HO) and micehomozygous for a rearranged heavy chain variable region nucleic acidsequence in the heavy chain loci and two human Vκ and five human Jκ genesegments in the light chain loci (MAID 1912HO 6032 HO; DLC x UHC).

FIG. 42 shows flow cytometric analyses of IgM surface expression on Bcells in harvested spleens from genetically modified control mice (VI3;1293HO 1460HO) and mice homozygous for a rearranged heavy chain variableregion nucleic acid sequence in the heavy chain loci and two human Vκand five human Jκ gene segments in the light chain loci (MAID 1912HO6032 HO; DLC x UHC). Cells were stained with fluorescent (PE-Cy7conjugated) antibody against IgM.

FIG. 43A shows the peripheral B cell development in genetically modifiedcontrol mice (VI3; 1293HO 1460HO) and mice homozygous for a rearrangedheavy chain variable region nucleic acid sequence in the heavy chainloci and two human Vκ and five human Jκ gene segments in the light chainloci (MAID 1912HO 6032 HO; DLC x UHC). The first (far left) contour plotshows CD93⁺ and B220⁺ splenocytes gated on CD19⁺ indicating immature andmature B cells. The second (middle) contour plot shows IgM⁺ and CD23⁺expression in immature B cells indicating T1 (IgD⁻IgM⁺CD21^(lo)CD23⁻),T2 (IgD^(hi)IgM^(hi)CD21^(mid)CD23⁺) and T3 B cell populations. Thethird (right) contour plot shows CD21⁺ (CD35⁺) and IgM⁺ expression ofmature B cells indicating first smaller populations which give rise tomarginal zone B cells and second larger populations which gives rise tofollicular (FO) B cells. Percentage of cells within each gated region isshown.

FIG. 43B shows the peripheral B cell development in genetically modifiedcontrol mice (VI3; 1293HO 1460HO) and mice homozygous for a rearrangedheavy chain variable region nucleic acid sequence in the heavy chainloci and two human Vκ and five human Jκ gene segments in the light chainloci (MAID 1912HO 6032 HO; DLC x UHC). The first (left) contour plotshows CD21⁺ (CD35⁺) and IgM⁺ expression of mature B cells indicating asmall population which give rise to marginal zone B cells and a secondpopulation which gives rise to follicular (FO) B cells. The second(middle) contour plot shows B220⁺ and CD23⁺ expression in mature B cellsindicating marginal zone (MZ) and marginal zone precursor(IgM^(hi)IgD^(hi)CD21^(hi)CD23⁺) B cell populations. The third (right)contour plot shows IgD⁺ and IgM⁺ expression in mature B cells indicatingFO-I (IgD^(hi)IgM^(int)CD21^(int)CD23⁺) and FO-II(IgD^(hi)IgM^(int)CD21^(int)CD23⁺) B cell populations. Percentage ofcells within each gated region is shown.

FIG. 44A shows representative contour plots of bone marrow stained for Band T cells (CD19⁺ and CD3⁺, respectively) from a genetically modifiedcontrol mouse (VI3; 1293HO 1460HO) and a mouse homozygous for arearranged heavy chain variable region nucleic acid sequence in theheavy chain loci and two human Vκ and five human Jκ gene segments in thelight chain loci (MAID 1912HO 6032 HO; DLC x UHC).

FIG. 44B shows the percentage of lymphocytes, total number ofcells/femur and number of CD19+ B cells in bone marrow harvested fromthe femurs of genetically modified control mice (VI3; 1293HO 1460HO) andmice homozygous for a rearranged heavy chain variable region nucleicacid sequence in the heavy chain loci and two human Vκ and five human Jκgene segments in the light chain loci (MAID 1912HO 6032 HO; DLC x UHC).

FIG. 45A shows representative contour plots of bone marrow gated onCD19⁺ and stained for ckit⁺ and CD43⁺ from a genetically modifiedcontrol mouse (VI3; 1293HO 1460HO) and a mouse homozygous for arearranged heavy chain variable region nucleic acid sequence in theheavy chain loci and two human Vκ and five human Jκ gene segments in thelight chain loci (MAID 1912HO 6032 HO; DLC x UHC). Pro and Pre B cellsare noted on the contour plots.

FIG. 45B shows the number of Pre (CD19⁺CD43⁻ckit⁻) and Pro(CD19⁺CD43⁺ckit⁺) B cells in bone marrow harvested from the femurs ofgenetically modified control mice (VI3; 1293HO 1460HO) and micehomozygous for a rearranged heavy chain variable region nucleic acidsequence in the heavy chain loci and two human Vκ and five human Jκ genesegments in the light chain loci (MAID 1912HO 6032HO; DLC x UHC).

FIG. 46A shows representative contour plots of bone marrow gated onsinglets stained for immunoglobulin M (IgM) and B220 from a geneticallymodified control mouse (VI3; 1293HO 1460HO) and a mouse homozygous for arearranged heavy chain variable region nucleic acid sequence in theheavy chain loci and two human Vκ and five human Jκ gene segments in thelight chain loci (MAID 1912HO 6032 HO; DLC x UHC). Immature, mature andpro/pre B cells are noted on each of the contour plots.

FIG. 46B shows the total number cell/femur, immature B (B220^(int)IgM⁺)and mature B (B220^(hi)IgM⁺) cells in bone marrow isolated from thefemurs of genetically modified control mice (VI3; 1293HO 1460HO) andmice homozygous for a rearranged heavy chain variable region nucleicacid sequence in the heavy chain loci and two human Vκ and five human Jκgene segments in the light chain loci (MAID 1912HO 6032 HO; DLC x UHC).

FIG. 47 shows representative contour plots of bone marrow gated onimmature (B220^(int)IgM⁺) and mature (B220^(hi)IgM⁺) B cells stained forIgλ and Igκ expression isolated from the femurs of a geneticallymodified control mouse (VI3; 1293HO 1460HO) and a mouse homozygous for arearranged heavy chain variable region nucleic acid sequence in theheavy chain loci and two human Vκ and five human Jκ gene segments in thelight chain loci (MAID 1912HO 6032 HO; DLC x UHC).

FIG. 48 shows the levels of antigen-specific mIgGs in the mouse sera(Wild type or 1912HO 6031 HET (homozygous DLC x heterozygous UHC))before footpad immunization, 23 days following a 1^(st) round of footpadimmunization, 5 weeks following the 1^(st) round of footpadimmunization, and after a 2^(nd) round of footpad immunization.

FIG. 49 illustrates the genomic structure of genetically modified F1mice containing a rearranged heavy chain variable region nucleic acidsequences in the kappa light chain loci (i.e., a rearranged heavy chainVDJ sequence operably linked to a kappa light chain constant nucleicacid sequence).

FIG. 50A in the top panel, shows representative contour plots of bonemarrow stained for B and T cells (CD19⁺ and CD3⁺, respectively) from awild type mouse (WT) and a mouse homozygous for a rearranged heavy chainvariable region nucleic acid sequence (hV_(H)3-23/D/J_(H)4) in the kappalight chain locus. The bottom panel shows representative contour plotsof bone marrow gated on CD19⁺ and stained for ckit⁺ and CD43⁺ from awild type mouse (WT) and a mouse homozygous for a rearranged heavy chainvariable region nucleic acid sequence (hV_(H)3-23/D/J_(H)4) in the kappalight chain locus. Pro and Pre B cells are noted on the contour plots ofthe bottom panel.

FIG. 50B shows the number of Pro (CD19⁺CD43⁺ckit⁺) and Pre(CD19⁺CD43⁻ckit⁻) B cells in bone marrow harvested from the femurs ofwild type mice (WT) and mice homozygous for a rearranged heavy chainvariable region nucleic acid sequence (hV_(H)3-23/D/J_(H)4) in the kappalight chain locus.

FIG. 51A shows representative contour plots of bone marrow gated onsinglets stained for immunoglobulin M (IgM) and B220 from a wild typemouse (WT) and a mouse homozygous for a rearranged heavy chain variableregion nucleic acid sequence (hV_(H)3-23/D/J_(H)4) in the kappa lightchain locus. Immature, mature and pro/pre B cells are noted on each ofthe contour plots.

FIG. 51B shows the total number of B (CD19⁺) and pro/pre B (IgM⁻B220⁺)cells in bone marrow isolated from the femurs of wild type mice (WT) andmice homozygous for a rearranged heavy chain variable region nucleicacid sequence (hV_(H)3-23/D/J_(H)4) in the kappa light chain locus.

FIG. 51C shows the number of immature B (B220^(int)IgM⁺) and mature B(B220^(hi)IgM⁺) cells in bone marrow isolated from the femurs of wildtype mice (WT) and mice homozygous for a rearranged heavy chain variableregion nucleic acid sequence (hV_(H)3-23/D/J_(H)4) in the kappa lightchain locus.

FIG. 52 shows representative contour plots of bone marrow gated onimmature (B220^(int)IgM⁺) and mature (B220^(hi)IgM⁺) B cells stained forIgλ and Igκ expression isolated from the femurs of wild type mice (WT)and mice homozygous for a rearranged heavy chain variable region nucleicacid sequence (hV_(H)3-23/D/J_(H)4) in the kappa light chain locus.

FIG. 53A, in the top panel, shows representative contour plots ofsplenocytes gated on singlets and stained for B and T cells (CD19⁺ andCD3⁺, respectively) from a wild type mouse (WT) and a mouse homozygousfor a rearranged heavy chain variable region nucleic acid sequence(hV_(H)3-23/D/J_(H)4) in the kappa light chain locus. The bottom panelshows representative contour plots of splenocytes gated on CD19⁺ andstained for immunoglobulin D (IgD) and immunoglobulin M (IgM) from awild type mouse (WT) and a mouse homozygous for a rearranged heavy chainvariable region nucleic acid sequence (hV_(H)3-23/D/J_(H)4) in the kappalight chain locus. Mature (56.9 for WT, 43 for hV_(H)3-23/D/J_(H)4 onkappa) and transitional (26.8 for WT, 34 for hV_(H)3-23/D/J_(H)4 onkappa) B cells are noted on each of the contour plots.

FIG. 53B shows the total number of CD19⁺ B cells, mature B cells(CD19⁺IgM^(lo)IgD^(hi)) and transitional B cells(CD19⁺IgM^(hi)IgD^(int)) in harvested spleens from wild type mice (WT)and mice homozygous a rearranged heavy chain variable region nucleicacid sequence (hV_(H)3-23/D/J_(H)4) in the kappa light chain locus.

FIG. 54A shows representative contour plots of Igλ⁺ and Igκ⁺ splenocytesgated on CD19⁺ from a wild type mouse (WT) and a rearranged heavy chainvariable region nucleic acid sequence (hV_(H)3-23/D/J_(H)4) in the kappalight chain locus.

FIG. 54B shows the total number of B cells (CD19⁺), Igκ⁺ B cells(CD19⁺Igκ⁺) and Igλ⁺ B cells (CD19⁺Igλ⁺) in harvested spleens from wildtype (WT) and mice homozygous for a rearranged heavy chain variableregion nucleic acid sequence (hV_(H)3-23/D/J_(H)4) in the kappa lightchain locus.

FIG. 55 shows the peripheral B cell development in the spleniccompartment of mice homozygous for a rearranged heavy chain variableregion nucleic acid sequence (hV_(H)3-23/D/J_(H)4) in the kappa lightchain locus compared to wild type mice. The first (left) contour plotshows CD93⁺ and B220⁺ splenocytes gated on CD19⁺ indicating immature andmature B cells. The second (middle) contour plot shows IgM⁺ and CD23⁺expression in immature B cells indicating T1, T2 and T3 B cellpopulations. The third (right) contour plot shows CD21⁺ (CD35⁺) and IgM⁺expression of mature B cells indicating a first smaller population thatgive rise to marginal zone B cells and a second larger population thatgives rise to follicular (FO) B cells. Percentage of cells within eachgated region is shown.

FIG. 56 illustrates the genomic structure of genetically modified F2mice homozygous for a rearranged heavy chain variable region nucleicacid sequence in the kappa light chain locus (MAID 6079HO; homozygous“UHC on kappa mouse”) and homozygous for a kappa light chain variableregion nucleic acid sequence in a heavy chain locus (MAID 1994HO; kappaon heavy (“KoH”) mouse).

FIG. 57A in the top panel, shows representative contour plots of bonemarrow stained for B and T cells (CD19⁺ and CD3⁺, respectively) from aVELOCIMMUNE® (VI3) and a mouse homozygous for a rearranged heavy chainvariable region nucleic acid sequence in the kappa light chain locus andhomozygous for a kappa light chain variable region nucleic acid sequencein a heavy chain locus. The bottom panel shows representative contourplots of bone marrow gated on CD19⁺ and stained for ckit⁺ and CD43⁺ froma VELOCIMMUNE® (VI3) and a mouse homozygous for a rearranged heavy chainvariable region nucleic acid sequence in the kappa light chain locus andhomozygous for a kappa light chain variable region nucleic acid sequencein a heavy chain locus. Pro and Pre B cells are noted on the contourplots of the bottom panel.

FIG. 57B shows the total number of B cells (CD19⁺) and the numbers ofPro (CD19⁺CD43⁺ckit⁺) and Pre (CD19⁺CD43⁻ckit⁻) B cells in bone marrowharvested from the femurs of VELOCIMMUNE® mice (1242HO 1640HO) and micehomozygous for a rearranged heavy chain variable region nucleic acidsequence in the kappa light chain locus and homozygous for a kappa lightchain variable region nucleic acid sequence in a heavy chain locus(1994HO 6079HO). Numbers are presented as both absolute number of cellsper femur and cell percentage.

FIG. 58A shows representative contour plots of bone marrow gated onsinglets stained for immunoglobulin M (IgM) and B220 from a VELOCIMMUNE®mouse (1242HO 1640HO) and a mouse homozygous for a rearranged heavychain variable region nucleic acid sequence in the kappa light chainlocus and homozygous for a kappa light chain variable region nucleicacid sequence in a heavy chain locus (1994HO 6079HO). Immature, matureand pro/pre B cells are noted on each of the contour plots.

FIG. 58B shows the number of immature B (B220^(int)IgM⁺) and mature B(B220^(int)IgM⁺) cells in bone marrow isolated from the femurs ofVELOCIMMUNE® mice (1242HO 1640HO) and mice homozygous for a rearrangedheavy chain variable region nucleic acid sequence in the kappa lightchain locus and homozygous for a kappa light chain variable regionnucleic acid sequence in a heavy chain locus (1994HO 6079HO). Numbersare presented as both absolute number of cells per femur and cellpercentage.

FIG. 59 shows representative contour plots of bone marrow gated onimmature (B220^(int)IgM⁺) and mature (B220^(hi)IgM⁺) B cells stained forIgλ and Igκ expression isolated from the femurs of VELOCIMMUNE® mice(1242HO 1640HO) and mice homozygous for a rearranged heavy chainvariable region nucleic acid sequence in the kappa light chain locus andhomozygous for a kappa light chain variable region nucleic acid sequencein a heavy chain locus (1994HO 6079HO).

FIG. 60A shows representative contour plots of splenocytes gated onCD19⁺ and stained for immunoglobulin D (IgD) and immunoglobulin M (IgM)from a VELOCIMMUNE® mouse (VI3; 1242HO 1640HO) and a mouse homozygousfor a rearranged heavy chain variable region nucleic acid sequence inthe kappa light chain locus and homozygous for a kappa light chainvariable region nucleic acid sequence in a heavy chain locus (1994HO6079HO). Mature and transitional/immature B cells are noted on each ofthe contour plots.

FIG. 60B shows the total number of CD19⁺ B cells, mature B cells(CD19⁺IgM^(lo)IgD^(hi)) and transitional B cells (CD19+IgM^(hi)IgD^(lo))in harvested spleens from VELOCIMMUNE® mice (1242HO 1640HO) and micehomozygous for a rearranged heavy chain variable region nucleic acidsequence in the kappa light chain locus and homozygous for a kappa lightchain variable region nucleic acid sequence in a heavy chain locus(1994HO 6079HO).

FIG. 61 shows the total number of B cells (CD19⁺), Igκ⁺ B cells(CD19⁺Igκ⁺) and Igλ⁺ B cells (CD19⁺Igλ⁺) in harvested spleens fromVELOCIMMUNE® mice (1242HO 1640HO) and mice homozygous for a rearrangedheavy chain variable region nucleic acid sequence in the kappa lightchain locus and homozygous for a kappa light chain variable regionnucleic acid sequence in a heavy chain locus (1994HO 6079HO). Numbersare presented as both absolute cell number and cell percentage oflymphocytes.

FIG. 62 shows the peripheral B cell development in the spleniccompartment of VELOCIMMUNE® mice (1242HO 1640HO) and mice homozygous fora rearranged heavy chain variable region nucleic acid sequence in thekappa light chain locus and homozygous for a kappa light chain variableregion nucleic acid sequence in a heavy chain locus (1994HO 6079HO). Thetop contour plot shows CD93⁺ and B220⁺ splenocytes gated on CD19⁺indicating immature and mature B cells. The bottom contour plot showsIgM⁺ and CD23⁺ expression in immature B cells indicating T1, T2 and T3 Bcell populations. Percentage of cells within each gated region is shown.

DETAILED DESCRIPTION

This invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention is defined bythe claims.

Unless defined otherwise, all terms and phrases used herein include themeanings that the terms and phrases have attained in the art, unless thecontrary is clearly indicated or clearly apparent from the context inwhich the term or phrase is used. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, particular methods andmaterials are now described. All publications mentioned are herebyincorporated by reference.

The term “antibody”, as used herein, includes immunoglobulin moleculescomprising four polypeptide chains, two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds. Each heavy chaincomprises a heavy chain variable domain and a heavy chain constantregion (C_(H)). The heavy chain constant region comprises three domains,C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises a light chainvariable domain and a light chain constant region (C_(L)). The heavychain and light chain variable domains can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDR), interspersed with regions that are more conserved, termedframework regions (FR). Each heavy and light chain variable domaincomprises three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2 andHCDR3; light chain CDRs may be abbreviated as LCDR1, LCDR2 and LCDR3).The term “high affinity” antibody refers to an antibody that has a K_(D)with respect to its target epitope about of 10⁻⁹ M or lower (e.g., about1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ M, or about 1×10⁻¹² M). In one embodiment,K_(D) is measured by surface plasmon resonance, e.g., BIACORE™; inanother embodiment, K_(D) is measured by ELISA.

The phrase “bispecific antibody” includes an antibody capable ofselectively binding two or more epitopes. Bispecific antibodiesgenerally comprise two nonidentical heavy chains, with each heavy chainspecifically binding a different epitope—either on two differentmolecules (e.g., different epitopes on two different immunogens) or onthe same molecule (e.g., different epitopes on the same immunogen). If abispecific antibody is capable of selectively binding two differentepitopes (a first epitope and a second epitope), the affinity of thefirst heavy chain for the first epitope will generally be at least oneto two or three or four or more orders of magnitude lower than theaffinity of the first heavy chain for the second epitope, and viceversa. Epitopes specifically bound by the bispecific antibody can be onthe same or a different target (e.g., on the same or a differentprotein). Exemplary bispecific antibodies include those with a firstheavy chain specific for a tumor antigen and a second heavy chainspecific for a cytotoxic marker, e.g., an Fc receptor (e.g., FcγRI,FcγRII, FcγRIII, etc.) or a T cell marker (e.g., CD3, CD28, etc.).Further, the second heavy chain variable domain can be substituted witha heavy chain variable domain having a different desired specificity.For example, a bispecific antibody with a first heavy chain specific fora tumor antigen and a second heavy chain specific for a toxin can bepaired so as to deliver a toxin (e.g., saporin, vinca alkaloid, etc.) toa tumor cell. Other exemplary bispecific antibodies include those with afirst heavy chain specific for an activating receptor (e.g., B cellreceptor, FcγRI, FcγRIIA, FcγRIIIA, FcαRI, T cell receptor, etc.) and asecond heavy chain specific for an inhibitory receptor (e.g., FcγRIIB,CD5, CD22, CD72, CD300a, etc.). Such bispecific antibodies can beconstructed for therapeutic conditions associated with cell activation(e.g. allergy and asthma). Bispecific antibodies can be made, forexample, by combining heavy chains that recognize different epitopes ofthe same immunogen. For example, nucleic acid sequences encoding heavychain variable sequences that recognize different epitopes of the sameimmunogen can be fused to nucleic acid sequences encoding the same ordifferent heavy chain constant regions, and such sequences can beexpressed in a cell that expresses an immunoglobulin light chain. Atypical bispecific antibody has two heavy chains each having three heavychain CDRs, followed by (N-terminal to C-terminal) a C_(H)1 domain, ahinge, a C_(H)2 domain, and a C_(H)3 domain, and an immunoglobulin lightchain that either does not confer epitope-binding specificity but thatcan associate with each heavy chain, or that can associate with eachheavy chain and that can bind one or more of the epitopes bound by theheavy chain epitope-binding regions, or that can associate with eachheavy chain and enable binding of one or both of the heavy chains to oneor both epitopes. Similarly, the term “trispecific antibody” includes anantibody capable of selectively binding three or more epitopes.

The term “cell” includes any cell that is suitable for expressing arecombinant nucleic acid sequence. Cells include those of prokaryotesand eukaryotes (single-cell or multiple-cell), bacterial cells (e.g.,strains of E. coli, Bacillus spp., Streptomyces spp., etc.),mycobacteria cells, fungal cells, yeast cells (e.g., S. cerevisiae, S.pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells(e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni,etc.), non-human animal cells, human cells, or cell fusions such as, forexample, hybridomas or quadromas. In some embodiments, the cell is ahuman, monkey, ape, hamster, rat, or mouse cell. In some embodiments,the cell is eukaryotic and is selected from the following cells: CHO(e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cell,Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK),HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21),Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cell, C127 cell,SP2/0, NS-0, MMT 060562, Sertoli cell, BRL 3A cell, HT1080 cell, myelomacell, tumor cell, and a cell line derived from an aforementioned cell.In some embodiments, the cell comprises one or more viral genes, e.g. aretinal cell that expresses a viral gene (e.g., a PER.C6™ cell).

The phrase “complementarity determining region,” or the term “CDR,”includes an amino acid sequence encoded by a nucleic acid sequence of anorganism's immunoglobulin genes that normally (i.e., in a wild-typeanimal) appears between two framework regions in a variable region of alight or a heavy chain of an immunoglobulin molecule (e.g., an antibodyor a T cell receptor). A CDR can be encoded by, for example, a germlinesequence or a rearranged or unrearranged sequence, and, for example, bya naive or a mature B cell or a T cell. A CDR can be somatically mutated(e.g., vary from a sequence encoded in an animal's germline), humanized,and/or modified with amino acid substitutions, additions, or deletions.In some circumstances (e.g., for a CDR3), CDRs can be encoded by two ormore sequences (e.g., germline sequences) that are not contiguous (e.g.,in an unrearranged nucleic acid sequence) but are contiguous in a B cellnucleic acid sequence, e.g., as the result of splicing or connecting thesequences (e.g., V-D-J recombination to form a heavy chain CDR3).

The term “conservative,” when used to describe a conservative amino acidsubstitution, includes substitution of an amino acid residue by anotheramino acid residue having a side chain R group with similar chemicalproperties (e.g., charge or hydrophobicity). In general, a conservativeamino acid substitution will not substantially change the functionalproperties of interest of a protein, for example, the ability of avariable region to specifically bind a target epitope with a desiredaffinity. Examples of groups of amino acids that have side chains withsimilar chemical properties include aliphatic side chains such asglycine, alanine, valine, leucine, and isoleucine; aliphatic-hydroxylside chains such as serine and threonine; amide-containing side chainssuch as asparagine and glutamine; aromatic side chains such asphenylalanine, tyrosine, and tryptophan; basic side chains such aslysine, arginine, and histidine; acidic side chains such as asparticacid and glutamic acid; and, sulfur-containing side chains such ascysteine and methionine. Conservative amino acids substitution groupsinclude, for example, valine/leucine/isoleucine, phenylalanine/tyrosine,lysine/arginine, alanine/valine, glutamate/aspartate, andasparagine/glutamine. In some embodiments, a conservative amino acidsubstitution can be substitution of any native residue in a protein withalanine, as used in, for example, alanine scanning mutagenesis. In someembodiments, a conservative substitution is made that has a positivevalue in the PAM250 log-likelihood matrix disclosed in Gonnet et al.(1992) Exhaustive Matching of the Entire Protein Sequence Database,Science 256:1443-45, hereby incorporated by reference. In someembodiments, the substitution is a moderately conservative substitutionwherein the substitution has a nonnegative value in the PAM250log-likelihood matrix.

In some embodiments, residue positions in an immunoglobulin light chainor heavy chain differ by one or more conservative amino acidsubstitutions. In some embodiments, residue positions in animmunoglobulin light chain or functional fragment thereof (e.g., afragment that allows expression and secretion from, e.g., a B cell) arenot identical to a light chain whose amino acid sequence is listedherein, but differs by one or more conservative amino acidsubstitutions.

The phrase “epitope-binding protein” includes a protein having at leastone CDR and that is capable of selectively recognizing an epitope, e.g.,is capable of binding an epitope with a K_(D) that is at about onemicromolar or lower (e.g., a K_(D) that is about 1×10⁻⁶ M, 1×10⁻⁷ M,1×10⁻⁹ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ M, or about 1×10⁻¹² M).Therapeutic epitope-binding proteins (e.g., therapeutic antibodies)frequently require a K_(D) that is in the nanomolar or the picomolarrange.

The phrase “functional fragment” includes fragments of epitope-bindingproteins that can be expressed, secreted, and specifically bind to anepitope with a K_(D) in the micromolar, nanomolar, or picomolar range.Specific recognition includes having a K_(D) that is at least in themicromolar range, the nanomolar range, or the picomolar range.

The term “germline” in reference to an immunoglobulin nucleic acidsequence includes a nucleic acid sequence that can be passed to progeny.

The phrase “heavy chain,” or “immunoglobulin heavy chain” includes animmunoglobulin heavy chain sequence, including immunoglobulin heavychain constant region sequence, from any organism. Heavy chain variabledomains include three heavy chain CDRs and four FR regions, unlessotherwise specified. Fragments of heavy chains include CDRs, CDRs andFRs, and combinations thereof. A typical heavy chain has, following thevariable domain (from N-terminal to C-terminal), a C_(H)1 domain, ahinge, a C_(H)2 domain, and a C_(H)3 domain. A functional fragment of aheavy chain includes a fragment that is capable of specificallyrecognizing an epitope (e.g., recognizing the epitope with a K_(D) inthe micromolar, nanomolar, or picomolar range), that is capable ofexpressing and secreting from a cell, and that comprises at least oneCDR. A heavy chain variable domain is encoded by a variable region genesequence, which generally comprises V_(H), D_(H), and J_(H) segmentsderived from a repertoire of V_(H), D_(H), and J_(H) segments present inthe germline. Sequences, locations and nomenclature for V, D, and Jheavy chain segments for various organisms can be found in IMGTdatabase, www.imgt.org.

The term “identity” when used in connection with a sequence, includesidentity as determined by a number of different algorithms known in theart that can be used to measure nucleotide and/or amino acid sequenceidentity. In some embodiments described herein, identities aredetermined using a ClustalW v. 1.83 (slow) alignment employing an opengap penalty of 10.0, an extend gap penalty of 0.1, and using a Gonnetsimilarity matrix (MACVECTOR™ 10.0.2, MacVector Inc., 2008). The lengthof the sequences compared with respect to identity of sequences willdepend upon the particular sequences, but in the case of a light chainconstant domain, the length should contain sequence of sufficient lengthto fold into a light chain constant domain that is capable ofself-association to form a canonical light chain constant domain, e.g.,capable of forming two beta sheets comprising beta strands and capableof interacting with at least one C_(H)1 domain of a human or a mouse. Inthe case of a C_(H)1 domain, the length of sequence should containsequence of sufficient length to fold into a C_(H)1 domain that iscapable of forming two beta sheets comprising beta strands and capableof interacting with at least one light chain constant domain of a mouseor a human.

The phrase “immunoglobulin molecule” includes two immunoglobulin heavychains and two immunoglobulin light chains. The heavy chains may beidentical or different, and the light chains may be identical ordifferent.

The phrase “light chain” includes an immunoglobulin light chain sequencefrom any organism, and unless otherwise specified includes human kappaand lambda light chains and a VpreB, as well as surrogate light chains.Light chain variable domains typically include three light chain CDRsand four framework (FR) regions, unless otherwise specified. Generally,a full-length light chain includes, from amino terminus to carboxylterminus, a variable domain that includesFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant region. Alight chain variable domain is encoded by a light chain variable regiongene sequence, which generally comprises V_(L) and J_(L) segments,derived from a repertoire of V and J segments present in the germline.Sequences, locations and nomenclature for V and J light chain segmentsfor various organisms can be found in IMGT database, www.imgt.org. Lightchains include those, e.g., that do not selectively bind either a firstor a second epitope selectively bound by the epitope-binding protein inwhich they appear. Light chains also include those that bind andrecognize, or assist the heavy chain with binding and recognizing, oneor more epitopes selectively bound by the epitope-binding protein inwhich they appear. Common or universal light chains include thosederived from a human Vκ1-39Jκ5 gene or a human Vκ3-20Jκ1 gene, andinclude somatically mutated (e.g., affinity matured) versions of thesame. Dual light chains (DLC) include those derived from a light chainlocus comprising no more than two human Vκ segments, e.g., a humanVκ1-39 gene segment and a human Vκ3-20 gene segment, and includesomatically mutated (e.g., affinity matured) versions of the same.

The phrase “somatically hypermutated” includes reference to a nucleicacid sequence from a B cell that has undergone class-switching, whereinthe nucleic acid sequence of an immunoglobulin variable region (e.g.,nucleotide sequence encoding a heavy chain variable domain or includinga heavy chain CDR or FR sequence) in the class-switched B cell is notidentical to the nucleic acid sequence in the B cell prior toclass-switching, such as, for example, a difference in a CDR orframework nucleic acid sequence between a B cell that has not undergoneclass-switching and a B cell that has undergone class-switching.“Somatically mutated” includes reference to nucleic acid sequences fromaffinity-matured B cells that are not identical to correspondingimmunoglobulin variable region sequences in B cells that are notaffinity-matured (i.e., sequences in the genome of germline cells). Thephrase “somatically mutated” also includes reference to animmunoglobulin variable region nucleic acid sequence from a B cell afterexposure of the B cell to an epitope of interest, wherein the nucleicacid sequence differs from the corresponding nucleic acid sequence priorto exposure of the B cell to the epitope of interest. The phrase“somatically mutated” refers to sequences from antibodies that have beengenerated in an animal, e.g., a mouse having human immunoglobulinvariable region nucleic acid sequences, in response to an immunogenchallenge, and that result from the selection processes inherentlyoperative in such an animal.

The term “unrearranged,” with reference to a nucleic acid sequence,includes nucleic acid sequences that exist in the germline of an animalcell.

The phrase “variable domain” includes an amino acid sequence of animmunoglobulin light or heavy chain (modified as desired) that comprisesthe following amino acid regions, in sequence from N-terminal toC-terminal (unless otherwise indicated): FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

The term “operably linked” refers to a relationship wherein thecomponents operably linked function in their intended manner. In oneinstance, a nucleic acid sequence encoding a protein may be operablylinked to regulatory sequences (e.g., promoter, enhancer, silencersequence, etc.) so as to retain proper transcriptional regulation. Inone instance, a nucleic acid sequence of an immunoglobulin variableregion (or V(D)J segments) may be operably linked to a nucleic acidsequence of an immunoglobulin constant region so as to allow properrecombination between the sequences into an immunoglobulin heavy orlight chain sequence.

“Functional” as used herein, e.g., in reference to a functionalpolypeptide, includes a polypeptide that retains at least one biologicalactivity normally associated with the native protein. In anotherinstance, a functional immunoglobulin gene segment may include avariable gene segment that is capable of productive rearrangement togenerate a rearranged immunoglobulin gene sequence.

“Neutral pH” includes pH between about 7.0 and about 8.0, e.g., pHbetween about 7.0 and about 7.4, e.g., between about 7.2 and about 7.4,e.g., physiological pH. “Acidic pH” includes pH of 6.0 or lower, e.g.,pH between about 5.0 and about 6.0, pH between about 5.75 and about 6.0,e.g., pH of endosomal or lysosomal compartments.

The term “polymorphic variant” as used herein includes a sequence inwhich one or more nucleotides or amino acids have been substituted by adifferent nucleotides or amino acid as compared to the given sequence.Polymorphic alleles of the human immunoglobulin heavy chain variablegene segments (V_(H) genes) have largely been the result ofinsertion/deletion of gene segments and single nucleotide differenceswithin coding regions, both of which have the potential to havefunctional consequences on the immunoglobulin molecule. Examples ofcommon polymorphic alleles of the human immunoglobulin V_(H) genes arewell known in the art (see, for example, U.S. Ser. No. 13/653,456,incorporated by reference herein in its entirety).

The term “substantial” or “substantially all” when used to refer to anamount of gene segments (e.g., “substantially all” V, D, or J genesegments) includes both functional and non-functional gene segments andincludes, in various embodiments, e.g., 80% or more, 85% or more, 90% ormore, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or moreof all V, D, or J gene segments. In various embodiments, “substantiallyall” gene segments include, e.g., at least 95%, 96%, 97%, 98%, or 99% offunctional (i.e., non-pseudogene) gene segments).

Non-Human Animals Comprising a Rearranged Heavy Chain Variable RegionGene Sequence and Optionally a Limited Repertoire of Unrearranged LightChain Variable Gene Segments

While a variety of bispecific antibodies with dual antigen bindingproperties have been developed, the specificity and affinity of thelight chain or heavy chain variable regions in conventional bispecificantibodies had to be sacrificed to some extent because, in conventionalbispecific antibodies, either a heavy chain or a light chain variableregion alone contributes to binding each separate antigenic determinant,whereas in regular antibodies, both light and heavy chain variableregions can contribute to binding the same antigenic determinant.

Therefore, generation of light chain variable regions that have anability to bind an antigen independently from a heavy chain variableregion can be useful for making light chain variable domains (V_(L)s)for use in antigen-binding molecules (e.g., bispecific binding moleculesthat comprise a heavy chain constant region (e.g., selected from aC_(H)1, a hinge, a C_(H)2, a C_(H)3, and a combination thereof) fusedwith V_(L)), particularly those that do not comprise a heavy chainvariable domain, including heterodimers having the same or similar heavychain constant region but V_(L)s with different specificities and/oraffinities.

One approach to produce such light chain variable domains that can bindto an antigen independently from a heavy chain variable region is toapply a selective pressure on nucleotide sequences that encode avariable region or domain of a light chain (V_(L)) to generate lightchain CDR3s with more diverse antigenic binding repertoire. As disclosedherein, this can be achieved by generating a genetically modifiednon-human animal that contains, in its genome, a rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence. Sincethe heavy chain sequence is restricted to a common or universal (i.e.,the same or a very similar) sequences in these animals, the light chainvariable region nucleotide sequences (i.e., genes) will be forced tomake light chain CDR3s with more diverse and efficient antigenic bindingproperties, which can bind an antigenic determinant independently fromheavy chain variable regions. Furthermore, as disclosed herein, theprecise replacement of germline variable region gene segments (e.g., byhomologous recombination-mediated gene targeting) allows for makinganimals (e.g., mice) that have partly human immunoglobulin loci. Becausethe partly human immunoglobulin loci rearrange, hypermutate, andsomatically mutate (e.g., class switch) normally, the partly humanimmunoglobulin loci generate antibodies in the animal that comprisehuman variable regions. These animals exhibit a humoral immune systemthat is substantially similar to wild type animals, and display normalcell populations and normal lymphoid organ structures—even where theanimals lack a full repertoire of human variable region gene segments.Immunizing these animals (e.g., mice) results in robust humoralresponses that display a wide diversity of variable gene segment usage.Nucleotide sequences that encode the variable regions can be identifiedand cloned, then fused (e.g., in an in vitro system) with any sequencesof choice, e.g., any immunoglobulin isotype suitable for a particularuse, resulting in an antibody or antigen-binding protein derived whollyfrom human sequences.

In addition, by utilizing animals (e.g., mice or rats) that have arestricted (limited) light chain variable region gene segmentrepertoire, e.g., a restricted light chain variable segment repertoirecomprising one or more but less than the wild type number of human V_(L)gene segments (e.g., a dual light chain or “DLC,” US Patent ApplicationPublication No. 2013/0198880, incorporated by reference herein in itsentirety) in combination with the rearranged human immunoglobulin heavychain variable region nucleotide sequence described above, animmunoglobulin light chain variable domain that can more efficientlypair with an immunoglobulin heavy chain variable domain can be produced.Furthermore, by introducing histidine codons, e.g., via addition of oneor more histidine codons or substitution of one or more non-histidinecodons with histidine codons, into the limited light chain variable genesegments in the genome of the non-human animals described herein, lightchain variable region amino acid sequences that can confer improvedpH-dependent recyclability to the antigen-binding proteins (e.g.,bispecific or trispecific antibodies) can be generated.

In some embodiments, the genetically modified non-human animals asdescribed herein provide a greater yield of antibodies, while limitingdiversity at the same time, thereby increasing the probability ofsuccessful pairing of light chains with heavy chains generated in anon-human animal comprising a single rearranged light chain variableregion (e.g., a Universal Light Chain (“ULC”) mouse; see, e.g., U.S.pre-grant publication 2013/0185821, incorporated by reference herein).In some embodiments, the light chains may themselves exhibitantigen-binding properties. In some embodiments, the non-human animalmay be induced to produce antigen-binding proteins exhibiting antigenspecificity that resides in their light chains (e.g., by limiting amouse or rat's immunoglobulin heavy chain repertoire; e.g., by replacingthe mouse or rat heavy chain locus with a locus comprising a singlerearranged human immunoglobulin heavy chain variable region nucleotidesequence). In some embodiments, antigen-binding proteins (e.g.,antibodies) produced in such animals will be specific for a particularfirst epitope (e.g., effector antigens, cytotoxic molecules, Fcreceptors, toxins, activating or inhibitory receptors, T cell markers,immunoglobulin transporters, etc.) through their light chain binding.Such epitope-specific human light chains derived from these non-humananimals may be co-expressed with human heavy chains derived from a mousewith a limited light chain repertoire, e.g., a ULC mouse or rat, whereinthe heavy chain is selected based on its ability to bind a secondepitope (e.g., a second epitope on a different antigen).

In various aspects, a non-human animal is provided comprising in itsgermline genome an immunoglobulin heavy chain locus that comprises arearranged human immunoglobulin heavy chain variable region nucleotidesequence (i.e., a rearranged heavy chain VDJ sequence). In someembodiments, the rearranged human immunoglobulin heavy chain variableregion nucleotide sequence is operably linked to a human or a non-humanheavy chain constant region sequence. In some embodiments, animmunoglobulin heavy chain variable domain encoded by the rearrangedheavy chain variable region nucleotide sequence is not immunogenic tothe non-human animal. In some embodiments, the non-human animal ismodified to comprise a nucleotide sequence that encodes two copies,three copies, four copies or more of the rearranged heavy chain variabledomain operably linked to a heavy chain constant domain. In someembodiments, the nucleotide sequence encodes a plurality of copies ofthe rearranged human immunoglobulin heavy chain variable regionnucleotide sequence. For example, the nucleotide sequence can encode atleast one, two, three, four, five copies of the rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence. In someembodiments, the nucleotide sequence encodes 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 copies of the rearranged human immunoglobulin heavy chain variableregion nucleotide sequence. In some embodiments, the locus comprises aplurality of copies of the rearranged human immunoglobulin heavy chainvariable region nucleotide sequence operably linked to a heavy chainconstant domain gene sequence.

In other aspects, a non-human animal is provided that is geneticallyengineered to contain an immunoglobulin light chain locus that encodes arearranged heavy chain variable domain (i.e., a light chain locus thatcomprises a rearranged human immunoglobulin heavy chain variable regionnucleotide sequence) operably linked to a human or a non-human lightchain constant region gene sequence. For example, in some embodiments, arearranged human immunoglobulin heavy chain variable region nucleotidesequence (i.e., a pre-designed VDJ region; i.e., a common or universalheavy chain sequence) can be operably linked to a light chain constantregion gene sequence by targeting the rearranged heavy chain sequenceinto a mouse or rat light chain loci, either kappa or lambda. Thus, insome embodiments, the nucleotide sequence encoding the rearranged heavychain variable domain is present in the germline genome of the non-humananimal. In some embodiments, the rearranged heavy chain variable domainexpressed by the genetically modified non-human animal is notimmunogenic to the non-human animal. In some embodiments, the non-humananimal is modified to comprise a nucleotide sequence that encodes twocopies, three copies, four copies or more of the rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence operablylinked to a light chain constant domain. In some embodiments, thenucleotide sequence can encode a plurality of copies of the rearrangedhuman immunoglobulin heavy chain variable region nucleotide sequence.For example, the nucleotide sequence encodes at least 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 copies of the rearranged human immunoglobulin heavy chainvariable region nucleotide sequence. In some embodiments, the locuscomprises a plurality of copies of the rearranged human immunoglobulinheavy chain variable region nucleotide sequence operably linked to alight chain constant domain gene sequence.

In various aspects, the immunoglobulin light chain locus of thenon-human animals described herein comprises a limited repertoire oflight chain variable gene segments, e.g., one or more but less than thewild type number of human V_(L) gene segments; and one or more humanJ_(L) gene segments, operably linked to a non-human light chain constantregion nucleic acid sequence. Thus, genetically modified non-humananimals are provided comprising in their genomes: (i) an immunoglobulinheavy chain locus that comprises a rearranged human heavy chain variableregion nucleic acid sequence operably linked to a human or non-humanheavy chain constant region nucleic acid sequence; and (ii) animmunoglobulin light chain locus comprising two or more but less thanthe wild type number of human immunoglobulin light chain variable V_(L)and J_(L) gene segments operably linked to a light chain constant regionnucleic acid sequence. In some embodiments, the light chain constantregion is a rat or a mouse constant region, e.g., a rat or a mouse Cκconstant region. In some embodiments, the human variable region genesegments are capable of rearranging and encoding human variable domainsof an antibody, and the non-human animal does not comprise an endogenousV_(L) gene segment. In some embodiments, the non-human animal comprisesfive human Jκ gene segments, e.g., Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5 genesegments. In some embodiments, the immunoglobulin light chain locuscomprises two human V_(L) gene segments, Vκ1-39 and Vκ3-20. In someembodiments, one or more (e.g., 2, 3, 4, or 5) human V_(L) gene segmentsand two or more human J_(L) gene segments are present at an endogenouslight chain locus, e.g., at an endogenous kappa light chain locus. Insome embodiments, the mouse comprises a functional λ light chain locus.In some embodiments, the mouse comprises a non-functional λ light chainlocus. In some embodiments, the one or more human V_(H), one or morehuman D_(H), and one or more human J_(H) gene segments are operablylinked to a mouse or a rat heavy chain constant region sequence.

In some embodiments, genetically modified mice comprising in theirgenomes (i) an immunoglobulin heavy chain locus that comprises arearranged human heavy chain variable region nucleic acid sequenceoperably linked to a human or non-human heavy chain constant regionnucleic acid sequence, and (ii) an immunoglobulin light chain locuscomprising two or more but less than the wild type number of humanimmunoglobulin light chain variable V_(L) and J_(L) gene segmentsoperably linked to a light chain constant region nucleic acid sequence,demonstrate CD19+ B cell numbers and mature B cell numbers that aresubstantially the same as the numbers observed in wild type mice or micecontaining other modifications of their immunoglobulin loci (i.e.,genetically modified control mice; e.g., VELOCIMMUNE® mice, in which thehumoral immune system of the mouse functions like that of a wild typemouse). In some embodiments, such mice demonstrate an increase inimmature B cell numbers in the spleen compared to genetically modifiedcontrol mice. In specific embodiments, such mice demonstrate about a2-fold, about a 3-fold, about a 4-fold, or about a 5-fold or greaterfold increase in immature B cell numbers in the spleen compared togenetically modified control mice. In some embodiments, such mice arealso substantially similar to wild type mice or genetically modifiedcontrol mice with respect to kappa and gamma light chain usage insplenic B cells. In some embodiments, such mice demonstrate increasedsurface IgM on splenic B cells (i.e., more IgM surface expression percell) as compared to genetically modified control mice. In someembodiments, such mice demonstrate altered peripheral B cell developmentthrough various stages of B cell development in the splenic compartmentcompared to genetically modified control mice, for example an increasein immature, T1 and/or marginal zone B cells. In some embodiments, suchmice demonstrate numbers of CD19+ B cells in the bone marrow compartmentthat are substantially similar to the numbers demonstrated ingenetically modified control mice. In some embodiments, such micedemonstrate fewer pro-B cells in the bone marrow compared to geneticallymodified control mice. In specific embodiments, the numbers of pro-Bcells in the bone marrow compartment are reduced by about 2-fold, about5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold ormore compared to genetically modified control mice. In some embodiments,such mice demonstrate about 2-fold, about 3-fold, about 4-fold, about5-fold, etc. fewer immature and/or mature B cells in the bone marrowcompared to genetically modified control mice. In some embodiments, suchmice exhibit a slight preference (e.g., 2-fold increase) in the bonemarrow compartment for usage of lambda light chain genes compared togenetically modified control mice.

In another aspect, a non-human animal is provided comprising agenetically modified immunoglobulin locus comprising: (a) a firstnucleotide sequence that encodes a rearranged heavy chain variabledomain (i.e., where the first nucleotide sequence is a rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence), whereinthe first nucleotide sequence is operably linked to a light chainconstant region gene sequence; and (b) a second nucleotide sequence thatencodes a human light chain variable domain (i.e., where the secondnucleotide sequence is an unrearranged human immunoglobulin light chainvariable region nucleotide sequence), wherein the second nucleotidesequence is operably linked to a heavy chain constant region genesequence. For example, in some embodiments, a rearranged heavy chainfrom a pre-designed VDJ region (i.e., a rearranged human immunoglobulinheavy chain variable region nucleotide sequence; i.e., a common oruniversal heavy chain sequence) can be operably linked to a light chainconstant region gene sequence by targeting the rearranged heavy chainsequence into a mouse light chain loci, either kappa or lambda. Thus, asin other embodiments, this genetically engineered immunoglobulin locusmay be present in the germline genome of the non-human animal.Genetically modified non-human animals comprising a human immunoglobulinlight chain variable region nucleotide sequences in operable linkagewith a heavy chain constant region gene sequences are described in U.S.pre-grant publication 2012/0096572, which is incorporated herein byreference. In some embodiments, the first nucleotide sequence thatencodes the rearranged heavy chain variable domain is operably linked toa κ light chain constant region gene sequence. In some embodiments, thefirst nucleotide sequence that encodes the rearranged heavy chainvariable domain is operably linked to a mouse or rat κ light chainconstant region gene sequence. In some embodiments, the first nucleotidesequence that encodes the rearranged heavy chain variable domain isoperably linked to a human κ light chain constant region gene sequence.In some embodiments, the first nucleotide sequence that encodes therearranged heavy chain variable domain is operably linked to a λ lightchain constant region gene sequence. In some embodiments, the firstnucleotide sequence that encodes the rearranged heavy chain variabledomain is operably linked to a mouse or rat λ light chain constantregion gene sequence. In some embodiments, the first nucleotide sequencethat encodes the rearranged heavy chain variable domain is operablylinked to a human λ light chain constant region gene sequence.

In some embodiments, a genetically modified mouse comprising animmunoglobulin light chain locus containing a rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence and animmunoglobulin heavy chain locus containing unrearranged humanimmunoglobulin light chain variable domain sequences (e.g., kappa lightchain genes) presents CD19+ and pre-B cell frequencies in the bonemarrow that are altered relative to a wild type mouse or a geneticallymodified mouse with other modifications at an immunoglobulin locus(i.e., genetically modified control mice; e.g., VELOCIMMUNE® mice, inwhich the humoral immune system of the mouse functions like that of awild type mouse). In specific embodiments, the CD19+ B cell and pre-Bcell numbers in the bone marrow are 2-fold lower, 3-fold lower, 4-foldlower or 5-fold lower compared to a wild type mouse or a geneticallymodified immunoglobulin locus control mouse. In specific embodiments,the number of immature B cells in the bone marrow is 2-fold less, 3-foldless, 4-fold less or 5-fold less compared to a wild type mouse or agenetically modified immunoglobulin locus control mouse. In someembodiments, a genetically modified mouse comprising an immunoglobulinlight chain locus containing a rearranged human immunoglobulin heavychain variable region nucleotide sequence and an immunoglobulin heavychain locus containing unrearranged human immunoglobulin light chainvariable domain sequences (e.g., kappa light chain genes) does notexpress or essentially does not express lambda light chain genes in thebone marrow cells. In some embodiments, a genetically modified mousecomprising an immunoglobulin light chain locus containing a rearrangedhuman immunoglobulin heavy chain variable region nucleotide sequence andan immunoglobulin heavy chain locus containing unrearranged humanimmunoglobulin light chain variable domain sequences (e.g., kappa lightchain genes) has reduced levels of splenic B cells compared to a wildtype mouse or a genetically modified immunoglobulin locus control mouse.In specific embodiments, the levels of splenic B cells and mature Bcells are 2-fold lower, 3-fold lower, 4-fold lower or 5-fold lowercompared to a wild type mouse or a genetically modified immunoglobulinlocus control mouse. In some embodiments, a genetically modified mousecomprising an immunoglobulin light chain locus containing a rearrangedhuman immunoglobulin heavy chain variable region nucleotide sequence andan immunoglobulin heavy chain locus containing unrearranged humanimmunoglobulin light chain variable domain sequences (e.g., kappa lightchain genes) does not express or essentially does not express lambdalight chain genes in splenic B cells. In specific embodiments, agenetically modified mouse comprising an immunoglobulin light chainlocus containing a rearranged human immunoglobulin heavy chain variableregion nucleotide sequence and an immunoglobulin heavy chain locuscontaining unrearranged human immunoglobulin light chain variable domainsequences (e.g., kappa light chain genes) has an increased frequency ofcells in the T1 phase in the spleen compared to a wild type mouse or agenetically modified immunoglobulin locus control mouse.

In some embodiments, the non-human animal is a mammal. Althoughembodiments employing a rearranged human heavy chain variable domain ina mouse (i.e., a mouse with an immunoglobulin locus comprising arearranged human immunoglobulin heavy chain variable region nucleotidesequence) are extensively discussed herein, other non-human animals thatcomprise a genetically modified immunoglobulin locus encoding arearranged human heavy chain variable domain are also provided. Suchnon-human animals include any of those which can be genetically modifiedto express the rearranged human immunoglobulin heavy chain variableregion nucleotide sequence as disclosed herein, including, e.g.,mammals, e.g., mouse, rat, rabbit, pig, bovine (e.g., cow, bull,buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g.,marmoset, rhesus monkey), etc. For example, for those non-human animalsfor which suitable genetically modifiable ES cells are not readilyavailable, other methods are employed to make a non-human animalcomprising the genetic modification. Such methods include, e.g.,modifying a non-ES cell genome (e.g., a fibroblast or an inducedpluripotent cell) and employing somatic cell nuclear transfer (SCNT) totransfer the genetically modified genome to a suitable cell, e.g., anenucleated oocyte, and gestating the modified cell (e.g., the modifiedoocyte) in a non-human animal under suitable conditions to form anembryo. Methods for modifying a non-human animal genome (e.g., a pig,cow, rodent, chicken, etc. genome) include, e.g., employing a zincfinger nuclease (ZFN) or a transcription activator-like effectornuclease (TALEN) to modify a genome to include a rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence.

In some embodiments, the non-human animal is a small mammal, e.g., ofthe superfamily Dipodoidea or Muroidea. In some embodiments, thegenetically modified animal is a rodent. In some embodiments, the rodentis selected from a mouse, a rat, and a hamster. In some embodiments, therodent is selected from the superfamily Muroidea. In some embodiments,the genetically modified animal is from a family selected fromCalomyscidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamster, NewWorld rats and mice, voles), Muridae (true mice and rats, gerbils, spinymice, crested rats), Nesomyidae (climbing mice, rock mice, with-tailedrats, Malagasy rats and mice), Platacanthomyidae (e.g., spiny dormice),and Spalacidae (e.g., mole rates, bamboo rats, and zokors). In aspecific embodiment, the genetically modified rodent is selected from atrue mouse or rat (family Muridae), a gerbil, a spiny mouse, and acrested rat. In some embodiments, the genetically modified mouse is froma member of the family Muridae. In some embodiments, the animal is arodent. In specific embodiments, the rodent is selected from a mouse anda rat. In some embodiments, the non-human animal is a mouse.

In some embodiments, the non-human animal is a rodent that is a mouse ofa C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN,C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6N, C57BL/6NJ, C57BL/10,C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In another embodiment, themouse is a 129 strain. In some embodiments, the 129 strain is selectedfrom the group consisting of 129P1, 129P2, 129P3, 129X1, 129S1 (e.g.,129S1/SV, 129S1/SvIm), 129S2, 129S4, 129S5, 129S9/SvEvH, 129S6(129/SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, e.g., Festing et al.(1999) Revised nomenclature for strain 129 mice, Mammalian Genome10:836, see also, Auerbach et al. (2000) Establishment and ChimeraAnalysis of 129/SvEv- and C57BL/6-Derived Mouse Embryonic Stem CellLines). In some embodiments, the genetically modified mouse is a mix ofan aforementioned 129 strain and an aforementioned C57BL strain (e.g., aC57BL/6 strain). In another embodiment, the mouse is a mix ofaforementioned 129 strains, or a mix of aforementioned C57BL/6 strains.In some embodiments, the 129 strain of the mix is a 129S6 (129/SvEvTac)strain. In another embodiment, the mouse is a mix of a 129/SvEv- and aC57BL/6-derived strain. In a specific embodiment, the mouse is a mix ofa 129/SvEv- and a C57BL/6-derived strain as described in Auerbach et al.2000 BioTechniques 29:1024-1032. In another embodiment, the mouse is aBALB strain, e.g., BALB/c strain. In another embodiment, the mouse is amix of a BALB strain (e.g., BALB/c strain) and another aforementionedstrain.

In some embodiments, the non-human animal is a rat. In some embodiments,the rat is selected from a Wistar rat, an LEA strain, a Sprague Dawleystrain, a Fischer strain, F344, F6, ACI, and Dark Agouti (DA). In someembodiments, the rat strain is a mix of two or more of a strain selectedfrom the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344,F6, ACI and Dark Agouti (DA).

In some embodiments, a genetically modified mouse comprising in itsgermline genome an immunoglobulin heavy chain locus that comprises arearranged human immunoglobulin heavy chain variable region nucleotidesequence generates splenic mature and immature B cell populations thatare essentially normal relative to a wild type mouse. In someembodiments, such a genetically modified mouse has a slight decrease inthe usage of light chain lambda gene sequences relative to wild type insplenic B cells. In specific embodiments, such a genetically modifiedmouse uses light chain lambda gene sequences with a 2-fold, 3-fold,4-fold or 5-fold lower frequency than wild type in splenic B cells. Insome embodiments, such a genetically modified mouse has a slightdecrease in T1 population splenic B cells and an increase in marginalzone splenic B cells relative to wild type. In some embodiments, such agenetically modified mouse has near normal B cell populations in thebone marrow. In some embodiments, such a genetically modified mouse useslambda gene sequences with a frequency that is half or less than half ofthe frequency that lambda gene sequences are used in wild type.

In various embodiments, as described herein, the rearranged heavy chainvariable domain (e.g., the rearranged human immunoglobulin heavy chainvariable region nucleotide sequence) is derived from a human V, D, and Jgene sequence or segment. In some embodiments, the rearranged heavychain variable domain is derived from a human germline V segment, ahuman germline D segment, and a human germline J segment. In someembodiments, the human V_(H) segment corresponds to observed variants inthe human population.

In various embodiments, as described herein, the human V gene segment isselected from the group consisting of V_(H)1-2, V_(H)1-3, V_(H)1-8,V_(H)1-18, V_(H)1-24, V_(H)1-45, V_(H)1-46, V_(H)1-58, V_(H)1-69,V_(H)2-5, V_(H)2-26, V_(H)2-70, V_(H)3-7, V_(H)3-9, V_(H)3-11,V_(H)3-13, V_(H)3-15, V_(H)3-16, V_(H)3-20, V_(H)3-21, V_(H)3-23,V_(H)3-30, V_(H)3-30-3, V_(H) 3-30-5, V_(H)3-33, V_(H)3-35, V_(H)3-38,V_(H)3-43, V_(H)3-48, V_(H)3-49, V_(H)3-53, V_(H)3-64, V_(H)3-66,V_(H)3-72, V_(H)3-73, V_(H)3-74, V_(H)4-4, V_(H)4-28, V_(H)4-30-1,V_(H)4-30-2, V_(H)4-30-4, V_(H)4-31, V_(H)4-34, V_(H)4-39, V_(H)4-59,V_(H)4-61, V_(H)5-51, V_(H)6-1, V_(H)7-4-1, V_(H)7-81, and a polymorphicvariant thereof. In some embodiments, the human V segment is V_(H)3-23or polymorphic variant thereof. In various embodiments, as describedherein, the human D gene segment is selected from the group consistingof D1-1, D1-7, D1-14, D1-20, D1-26, D2-2, D2-8, D2-15, D2-21, D3-3,D3-9, D3-10, D3-16, D3-22, D4-4, D4-11, D4-17, D4-23, D5-12, D5-5,D5-18, D5-24, D6-6, D6-13, D6-19, D6-25, D7-27, and a polymorphicvariant thereof. In some embodiments, the human or non-human animalheavy chain constant region sequence comprises a sequence selected froma C_(H)1, a hinge, a C_(H)2, a C_(H)3, and a combination thereof. Inspecific embodiments, the constant region sequence comprises a C_(H)1, ahinge, a C_(H)2, and a C_(H)3. In various embodiments, as describedherein, the human J gene segment is selected from the group consistingof J_(H)1, J_(H)2, J_(H)3, J_(H)4, J_(H)S, J_(H)6, and a polymorphicvariant thereof. In some embodiments, the rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence encodesthe sequence of human V_(H)3-23/GY/J_(H)4-4 (SEQ ID NO: 137). In someembodiments, the rearranged heavy chain variable domain encoded by andexpressed from the rearranged human immunoglobulin heavy chain variableregion nucleotide sequence comprises the sequence of humanV_(H)3-23/X₁X₂/J (wherein X1 is any amino acid, and X2 is any aminoacid). In some embodiments, X₁ is Gly and X₂ is Tyr. In someembodiments, the rearranged heavy chain variable domain comprises thesequence of human V_(H)3-23/X₁X₂/J_(H)4-4 (wherein X1 is any amino acid,and X₂ is any amino acid). In some embodiments, X₂ is an amino acidcomprising a phenyl group. In specific embodiments, X₂ is selected fromTyr and Phe.

In some embodiments, the rearranged human immunoglobulin heavy chainvariable region nucleotide sequence comprises a human D segment that isnot autoreactive (non-immunogenic) in the animal. In some embodiments,the nucleotide sequence comprises a human D segment that is capable ofbeing expressed in a heavy chain variable sequence of a mature B cell ofa mouse. In some embodiments, the D segment is a segment that has beenexpressed in a mouse that comprises a humanized immunoglobulin locuscomprising a human V_(H), a human D, and a human J_(H) segment.

Various embodiments utilize or encompass features or sequenceinformation derived from VELOCIMMUNE® humanized mice. VELOCIMMUNE®humanized mice contain a precise, large-scale replacement of germlinevariable regions of mouse immunoglobulin heavy chain (IgH) andimmunoglobulin light chain (e.g., κ light chain, Igκ) with correspondinghuman immunoglobulin variable regions, at the endogenous loci (see,e.g., U.S. Pat. Nos. 6,596,541 and 8,502,018, the entire contents ofwhich are incorporated herein by reference). In total, about sixmegabases of mouse loci are replaced with about 1.5 megabases of humangenomic sequence. This precise replacement results in a mouse withhybrid immunoglobulin loci that make heavy and light chains that have ahuman variable regions and a mouse constant region. The precisereplacement of mouse V_(H)-D-J_(H) and Vκ-Jκ segments leave flankingmouse sequences intact and functional at the hybrid immunoglobulin loci.The humoral immune system of the mouse functions like that of a wildtype mouse. B cell development is unhindered in any significant respectand a rich diversity of human variable regions is generated in the mouseupon antigen challenge. Moreover, VELOCIMMUNE® humanized mice display anessentially normal, wild-type response to immunization that differs onlyin one significant respect from wild type mice—the variable regionsgenerated in response to immunization are fully human. VELOCIMMUNE®humanized mice are possible because immunoglobulin gene segments forheavy and κ light chains rearrange similarly in humans and mice.Although the loci are not identical, they are similar enough thathumanization of the heavy chain variable gene locus can be accomplishedby replacing about three million base pairs of contiguous mouse sequencethat contains all the V_(H), D, and J_(H) gene segments with about onemillion bases of contiguous human genomic sequence covering basicallythe equivalent sequence from a human immunoglobulin locus. For example,in some embodiments, the D segment is derived from a heavy chainexpressed in a mature B cell of a VELOCIMMUNE® humanized mouse immunizedwith an antigen, wherein the D segment contributes no more than twoamino acids to the heavy chain CDR3 sequence.

In particular embodiments, a VELOCIMMUNE® mouse comprising animmunoglobulin heavy chain locus encoding a rearranged heavy chainvariable domain (i.e., comprising an immunoglobulin heavy chain locusthat comprises a rearranged human immunoglobulin heavy chain variableregion nucleotide sequence) is provided. A VELOCIMMUNE® mouse somodified comprises a replacement of mouse immunoglobulin heavy chainvariable gene segments with a rearranged human immunoglobulin heavychain variable region nucleotide sequence (i.e., a Universal Heavy Chainsequence at an endogenous heavy chain locus), and a replacement of mouseimmunoglobulin κ light chain variable gene segments with at least 40human Vκ gene segments and five human Jκ gene segments. In someembodiments, the human Vκ gene segments are selected from the groupconsisting of Vκ1-5, Vκ1-6, Vκ1-8, Vκ1-9, Vκ1-12, Vκ1-13, Vκ1-16,Vκ1-17, Vκ1-22, Vκ1-27, Vκ1-32, Vκ1-33, Vκ1-35, Vκ1-37, Vκ1-39, Vκ1D-8,Vκ1D-12, Vκ1D-13, Vκ1D-16, Vκ1D-17, Vκ1D-22, Vκ1 D-27, Vκ1D-32, Vκ1D-33,Vκ1D-35, Vκ1D-37, Vκ1D-39, Vκ1D-42, Vκ1D-43, Vκ1-NL1, Vκ2-4, Vκ2-10,Vκ2-14, Vκ2-18, Vκ2-19, Vκ2-23, Vκ2-24, Vκ2-26, Vκ2-28, Vκ2-29, Vκ2-30,Vκ2-36, Vκ2-38, Vκ2-40, Vκ2D-10, Vκ2D-14, Vκ2D-18, Vκ2D-19, Vκ2D-23,Vκ2D-24, Vκ2D-26, Vκ2D- 28, Vκ2D-29, Vκ2D-30, Vκ2D-36, Vκ2D-38, Vκ2D-40,Vκ3-7, V_(κ)3-11, Vκ3-15, Vκ3-20, Vκ3-25, Vκ3-31, Vκ3-34, Vκ3D-7,Vκ3D-7, Vκ3D-11, Vκ3D-15, Vκ3D-15, Vκ3D-20, Vκ3D-25, Vκ3D-31, Vκ3D-34,Vκ3-NL1, Vκ3-NL2, Vκ3-NL3, Vκ3-NL4, Vκ3-NL5, Vκ4-1, Vκ5-2, Vκ6-21, \MD-21, Vκ6D-41, and Vκ7-3. In some embodiments, the human Vκ genesegments comprise Vκ4-1, Vκ5-2, Vκ7-3, Vκ2-4, Vκ1-5, and Vκ1-6. In oneembodiment, the Vκ gene segments comprise Vκ3-7, Vκ1-8, Vκ1-9, Vκ2-10,Vκ3-11, Vκ1-12, Vκ1-13, Vκ2-14, Vκ3-15 and Vκ1-16. In some embodiments,the human Vκ gene segments comprise Vκ1-17, Vκ2-18, Vκ2-19, Vκ3-20,Vκ6-21, Vκ1-22, Vκ1-23, Vκ2-24, Vκ3-25, Vκ2-26, Vκ1-27, Vκ2-28, Vκ2-29,and Vκ2-30. In some embodiments, the human Vκ gene segments compriseVκ3-31, Vκ1-32, Vκ1-33, Vκ3-34, Vκ1-35, Vκ2-36, Vκ1-37, Vκ2-38, Vκ1-39,and Vκ2-40. In specific embodiments, the Vκ gene segments comprisecontiguous human immunoglobulin κ gene segments spanning the humanimmunoglobulin κ light chain locus from Vκ4-1 through Vκ2-40, and the Jκgene segments comprise contiguous gene segments spanning the humanimmunoglobulin κ light chain locus from Jκ1 through Jκ5. In someembodiments, the rearranged human heavy chain variable domain nucleotidesequence is operably linked to a mouse heavy chain constant regionsequence. A VELOCIMMUNE® mouse comprising an immunoglobulin heavy chainlocus encoding a rearranged heavy chain variable domain (i.e.,comprising an immunoglobulin heavy chain locus that comprises arearranged human immunoglobulin heavy chain variable region nucleotidesequence) can be used in any of the aspects, embodiments, methods, etc.described herein.

In various embodiments, the rearranged human immunoglobulin heavy chainvariable region nucleotide sequence is operably linked to a human ormouse heavy chain constant region gene sequence (e.g., a heavy chainconstant region gene sequence that encodes an immunoglobulin isotypeselected from IgM, IgD, IgA, IgE, IgG, and combinations thereof). Forexample, genetically modified non-human animals are provided comprisingimmunoglobulin loci in which: (a) a first nucleotide sequence encodes arearranged heavy chain variable domain (i.e., where the first nucleotidesequence is a rearranged human immunoglobulin heavy chain variableregion nucleotide sequence), wherein the first nucleotide sequence isoperably linked to a human or non-human heavy chain constant region genesequence; and (b) a second nucleotide sequence encodes a light chainvariable domain (i.e., where the second nucleotide sequence is anunrearranged human immunoglobulin light chain variable nucleotidesequence), wherein the second nucleotide sequence is operably linked toa human or non-human light chain constant region gene sequence. In someembodiments, the human heavy chain constant region gene sequence isselected from a C_(H)1, a hinge, a C_(H)2, a C_(H)3, and combinationsthereof. In some embodiments, a mouse heavy chain constant region genesequence is selected from a C_(H)1, a hinge, a C_(H)2, a C_(H)3, andcombinations thereof. In some embodiments, further replacement ofcertain non-human animal constant region gene sequences with human genesequences (e.g., replacement of mouse C_(H)1 sequence with human C_(H)1sequence, and replacement of mouse C_(L) sequence with human C_(L)sequence) results in genetically modified non-human animals with hybridimmunoglobulin loci that make antibodies that have human variableregions and partly human constant regions, suitable for, e.g., makingfully human antibody fragments, e.g., fully human Fab's. In someembodiments, the rearranged human immunoglobulin heavy chain variableregion nucleotide sequence is operably linked to a rat heavy chainconstant region gene sequence. In some embodiments, the rat heavy chainconstant region gene sequence is selected from a C_(H)1, a hinge, aC_(H)2, a C_(H)3, and combinations thereof. In various embodiments, thegenetically modified immunoglobulin heavy chain locus of the non-humananimal comprises two copies, three copies, four copies or more of therearranged human immunoglobulin heavy chain variable region nucleotidesequence operably linked to a heavy chain constant domain gene sequence.In particular embodiments, the locus comprises a plurality of copies ofthe rearranged human immunoglobulin heavy chain variable regionnucleotide sequence operably linked to a heavy chain constant domaingene sequence.

In various embodiments, the heavy chain constant region nucleotidesequence comprises a modification in a C_(H)2 or a C_(H)3, wherein themodification increases the affinity of the heavy chain constant regionamino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0). In someembodiments, the heavy chain constant region nucleotide sequence encodesa human heavy chain constant region amino acid sequence comprising amodification at position 250 by EU numbering (263 by Kabat numbering)(e.g., E or Q); 250 by EU numbering (263 by Kabat numbering) and 428 byEU numbering (459 by Kabat numbering) (e.g., L or F); 252 by EUnumbering (265 by Kabat numbering) (e.g., L/Y/F/W or T), 254 by EUnumbering (267 by Kabat numbering) (e.g., S or T), and 256 by EUnumbering (269 by Kabat numbering)(e.g., S/R/Q/E/D or T); or amodification at position 428 by EU numbering (459 by Kabat numbering)and/or 433 by EU numbering (464 by Kabat numbering) (e.g., L/R/S/P/Q orK) and/or 434 by EU numbering (465 by Kabat numbering) (e.g., H/F or Y);or a modification at position 250 by EU numbering (263 by Kabatnumbering) and/or 428 by EU numbering (459 by Kabat numbering); or amodification at position 307 by EU numbering (326 by Kabat numbering) or308 by EU numbering (327 by Kabat numbering) (e.g., 308F, V308F), and434 by EU numbering (465 by Kabat numbering). In one embodiment, themodification comprises a 428L (e.g., M428L) and 434S (e.g., N434S)modification by EU numbering (a 459, e.g., M459L, and 465S (e.g., N465S)modification by Kabat numbering); a 428L, 259I (e.g., V259I), and 308F(e.g., V308F) modification by EU numbering (a 459L, 272I (e.g., V272I),and 327F (e.g., V327F) modification by Kabat numbering; a 433K (e.g.,H433K) and a 434 (e.g., 434Y) modification by EU numbering (a 464K(e.g., H464K) and a 465 (e.g., 465Y) modification by Kabat numbering; a252, 254, and 256 (e.g., 252Y, 254T, and 256E) modification by EUnumbering (a 265, 267, 269 (e.g., 265Y, 267T, and 269E) modification byKabat numbering; a 250Q and 428L modification (e.g., T250Q and M428L) byEU numbering (a 263Q and 459L modification, e.g., T263Q and M459L, byKabat numbering); and a 307 and/or 308 modification (e.g., 307F or 308P)by EU numbering (326 and/or 327 modification, e.g., 326F or 308P, byKabat numbering), wherein the modification increases the affinity of theheavy chain constant region amino acid sequence to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0). In some embodiments, the heavy chain constant regionnucleotide sequence encodes a human C_(H)2 amino acid sequencecomprising at least one modification between amino acid residues atpositions 252 and 257 by EU numbering (i.e., at least one modificationbetween amino acid positions 265 and 270 by Kabat numbering), whereinthe modification increases the affinity of the human C_(H)2 amino acidsequence to FcRn in an acidic environment (e.g., in an endosome where pHranges from about 5.5 to about 6.0). In some embodiments, the heavychain constant region nucleotide sequence encodes a human C_(H)2 aminoacid sequence comprising at least one modification between amino acidresidues at positions 307 and 311 (i.e., at least one modificationbetween amino acid positions 326 and 330 by Kabat numbering), whereinthe modification increases the affinity of the C_(H)2 amino acidsequence to FcRn in an acidic environment (e.g., in an endosome where pHranges from about 5.5 to about 6.0). In some embodiments, the heavychain constant region nucleotide sequence encodes a human C_(H)3 aminoacid sequence, wherein the C_(H)3 amino acid sequence comprises at leastone modification between amino acid residues at positions 433 and 436 byEU numbering (i.e., at least one modification between amino acidresidues at positions 464 and 467 by Kabat numbering), wherein themodification increases the affinity of the C_(H)3 amino acid sequence toFcRn in an acidic environment (e.g., in an endosome where pH ranges fromabout 5.5 to about 6.0). In some embodiments, the heavy chain constantregion nucleotide sequence encodes a human heavy chain constant regionamino acid sequence comprising a mutation selected from the groupconsisting of M428L by EU numbering (459 by Kabat numbering), N434S byEU numbering (465 by Kabat numbering), and a combination thereof. Insome embodiments, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L by EUnumbering (M459L by Kabat numbering), V259I by EU numbering (V272I byKabat numbering), V308F by EU numbering (V327 by Kabat numbering), and acombination thereof. In some embodiments, the heavy chain constantregion nucleotide sequence encodes a human heavy chain constant regionamino acid sequence comprising an N434A mutation by EU numbering (anN465A mutation by Kabat numbering). In some embodiments, the heavy chainconstant region nucleotide sequence encodes a human heavy chain constantregion amino acid sequence comprising a mutation selected from the groupconsisting of M252Y by EU numbering (M265Y by Kabat numbering), S254T byEU numbering (S267T by Kabat numbering), T256E by EU numbering (T269E byKabat numbering), and a combination thereof. In some embodiments, theheavy chain constant region nucleotide sequence encodes a human heavychain constant region amino acid sequence comprising a mutation selectedfrom the group consisting of T250Q by EU numbering (T263Q by Kabatnumbering), M428L by EU numbering (M459L by Kabat numbering), or both.In some embodiments, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of H433K by EUnumbering (H464K by Kabat numbering), N434Y by EU numbering (N465Y byKabat numbering), or both.

In some embodiments, a genetically modified immunoglobulin locuscomprises: (1) a first allele, wherein the rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence asdescribed herein is operably linked to a first heavy chain constantregion nucleotide sequence encoding a first CH₃ amino acid sequence of ahuman IgG selected from IgG1, IgG2, IgG4, and a combination thereof; and(2) a second allele, wherein the rearranged human immunoglobulin heavychain variable region nucleotide sequence as described herein isoperably linked to a second heavy chain constant region nucleotidesequence encoding a second C_(H)3 amino acid sequence of the human IgGselected from IgG1, IgG2, IgG4, and a combination thereof, and whereinthe second CH₃ amino acid sequence comprises a modification that reducesor eliminates binding for the second CH₃ amino acid sequence to ProteinA (see, for example, U.S. Pat. No. 8,586,713, which is incorporated byreference herein in its entirety). In some embodiments, the second CH₃amino acid sequence comprises an H95R modification (by IMGT exonnumbering; H435R by EU numbering). In one embodiment the second CH₃amino acid sequence further comprises an Y96F modification (by IMGT exonnumbering; H436F by EU). In another embodiment, the second CH₃ aminoacid sequence comprises both an H95R modification (by IMGT exonnumbering; H435R by EU numbering) and an Y96F modification (by IMGT exonnumbering; H436F by EU). In some embodiments, the second CH₃ amino acidsequence is from a modified human IgG1 and further comprises a mutationselected from the group consisting of D16E, L18M, N44S, K52N, V57M, andV82I (IMGT; D356E, L38M, N384S, K392N, V397M, and V422I by EU). In someembodiments, the second CH₃ amino acid sequence is from a modified humanIgG2 and further comprises a mutation selected from the group consistingof N44S, K52N, and V82I (IMGT: N384S, K392N, and V422I by EU). In someembodiments, the second CH₃ amino acid sequence is from a modified humanIgG4 and further comprises a mutation selected from the group consistingof Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (IMGT: Q355R, N384S,K392N, V397M, R409K, E419Q, and V422I by EU). In some embodiments, theheavy chain constant region amino acid sequence is a non-human constantregion amino acid sequence, and the heavy chain constant region aminoacid sequence comprises one or more of any of the types of modificationsdescribed above.

In various embodiments, Fc domains are modified to have altered Fcreceptor binding, which in turn affects effector function. In someembodiments, an engineered heavy chain constant region (C_(H)), whichincludes the Fc domain, is chimeric. As such, a chimeric C_(H) regioncombines C_(H) domains derived from more than one immunoglobulinisotype. For example, a chimeric C_(H) region comprises part or all of aC_(H)2 domain derived from a human IgG1, human IgG2 or human IgG4molecule, combined with part or all of a C_(H)3 domain derived from ahuman IgG1, human IgG2 or human IgG4 molecule. In some embodiments, achimeric C_(H) region contain a chimeric hinge region. For example, achimeric hinge may comprise an “upper hinge” amino acid sequence (aminoacid residues from positions 216 to 227 according to EU numbering; aminoacid residues from positions 226 to 240 according to Kabat numbering)derived from a human IgG1, a human IgG2 or a human IgG4 hinge region,combined with a “lower hinge” sequence (amino acid residues frompositions 228 to 236 according to EU numbering; amino acid positionsfrom positions 241 to 249 according to Kabat numbering) derived from ahuman IgG1, a human IgG2 or a human IgG4 hinge region. In someembodiments, the chimeric hinge region comprises amino acid residuesderived from a human IgG1 or a human IgG4 upper hinge and amino acidresidues derived from a human IgG2 lower hinge.

In some embodiments, the Fc domain may be engineered to activate all,some, or none of the normal Fc effector functions, without affecting theFc-containing protein's (e.g. antibody's) desired pharmacokineticproperties. For examples of proteins comprising chimeric C_(H) regionsand having altered effector functions, see U.S. Provisional ApplicationNo. 61/759,578, filed Feb. 1, 2013, which is herein incorporated in itsentirety.

In various aspects, the genome of the non-human animals is modified (i)to delete or render nonfunctional (e.g., via insertion of a nucleotidesequence (e.g., an exogenous nucleotide sequence)) in the immunoglobulinlocus or via non-functional rearrangement or inversion of all, orsubstantially all, endogenous functional immunoglobulin V_(H), D, J_(H)segments; and (ii) to comprise a rearranged human immunoglobulin heavychain variable region nucleotide sequence, wherein the nucleotidesequence is present at an endogenous locus (i.e., where the nucleotidesequence is located in a wild type non-human animal). In someembodiments, the rearranged human immunoglobulin heavy chain variableregion nucleotide sequence is at an ectopic locus in the genome (e.g.,at a locus different from the endogenous immunoglobulin chain locus inits genome, or within its endogenous locus, e.g., within animmunoglobulin variable locus, wherein the endogenous locus is placed ormoved to a different location in the genome). In some embodiments, e.g.,about 80% or more, about 85% or more, about 90% or more, about 95% ormore, about 96% or more, about 97% or more, about 98% or more, or about99% or more of all endogenous functional heavy chain V, D, or J genesegments are deleted or rendered non-functional. In some embodiments,e.g., at least 95%, 96%, 97%, 98%, or 99% of endogenous functional heavychain V, D, or J gene segments are deleted or rendered non-functional.In some embodiments, the rearranged human immunoglobulin heavy chainvariable region nucleotide sequence is operably linked to a human ornon-human heavy chain constant region gene sequence. In someembodiments, the rearranged human immunoglobulin heavy chain variableregion nucleotide sequence is operably linked to a human or non-humanlight chain constant region gene sequence, either kappa or lambda.

In some embodiments, the genetically modified non-human animal comprisesa modification that deletes or renders non-functional endogenousfunctional V_(H), D, and J_(H) heavy chain variable gene segments andendogenous functional light chain variable V_(L) and J_(L) genesegments; and comprises (i) a rearranged human immunoglobulin heavychain variable region nucleotide sequence and (ii) a nucleotide sequenceencoding unrearranged human immunoglobulin light chain V gene segments(V_(L)) and unrearranged human immunoglobulin light chain J genesegments (J_(L)) (i.e., where the nucleotide sequence is located in awild-type non-human animal) or at an ectopic location (e.g., at a locusdifferent from the endogenous immunoglobulin chain locus in its genome,or within its endogenous locus, e.g., within an immunoglobulin variableregion locus, wherein the endogenous locus is placed or moved to adifferent location in the genome). In some embodiments, the geneticallymodified non-human animal comprises a modification that deletes orrenders non-functional endogenous functional V_(H), D, and J_(H) heavychain variable gene segments and endogenous functional light chainvariable V_(L) and J_(L) gene segments; and comprises (i) a rearrangedhuman immunoglobulin heavy chain variable region nucleotide sequence and(ii) one or more but less than the wild type number of humanimmunoglobulin light chain variable region gene segments (V_(L) andJ_(L)) at an endogenous location (i.e., where the nucleotide sequence islocated in a wild-type non-human animal) or at an ectopic location(e.g., at a locus different from the endogenous immunoglobulin chainlocus in its genome, or within its endogenous locus, e.g., within animmunoglobulin variable region locus, wherein the endogenous locus isplaced or moved to a different location in the genome). In someembodiments, e.g., about 80% or more, about 85% or more, about 90% ormore, about 95% or more, about 96% or more, about 97% or more, about 98%or more, or about 99% or more of all endogenous functional heavy chainV, D, or J gene segments are deleted or rendered non-functional. In someembodiments, e.g., at least 95%, 96%, 97%, 98%, or 99% of endogenousfunctional heavy chain V, D, or J gene segments are deleted or renderednon-functional. In some embodiments, the rearranged human immunoglobulinheavy chain variable region nucleotide sequence is operably linked to ahuman or non-human heavy chain constant region gene sequence. In someembodiments, the rearranged human immunoglobulin heavy chain variableregion nucleotide sequence is operably linked to a human or non-humanlight chain constant region gene sequence, either kappa or lambda.

Various embodiments encompass light chain variable domains. Nucleic acidsequences encoding light chain variable domains may be used in makingthe genetically modified non-humans described herein, may be expressedby such animals, and/or may encode amino acids present in antibodiesbodied produced by (or derived from sequences diversified by) suchanimals. In some embodiments, the light chain variable domain is a humanκ light chain variable domain. In some embodiments, the light chainvariable domain is a mouse κ light chain variable domain. In someembodiments, the light chain variable domain is a rat κ light chainvariable domain. In some embodiments, the light chain variable domain isa human λ light chain variable domain. In some embodiments, the lightchain variable domain is a mouse λ light chain variable domain. In someembodiments, the light chain variable domain is a rat λ light chainvariable domain.

In various embodiments, the light chain variable domains produced by thegenetically modified non-human animals described herein are encoded byone or more mouse or human immunoglobulin κ light chain variable genesegments. In some embodiments, the one or more mouse immunoglobulin κlight chain variable gene segments comprises about three megabases ofthe mouse immunoglobulin κ light chain locus. In some embodiments, theone or more mouse immunoglobulin κ light chain variable gene segmentscomprises at least 137 Vκ gene segments, at least five Jκ gene segmentsor a combination thereof of the mouse immunoglobulin κ light chainlocus. In some embodiments, the one or more human immunoglobulin κ lightchain variable gene segments comprises about one-half megabase of ahuman immunoglobulin κ light chain locus. In specific embodiments, theone or more human immunoglobulin κ light chain variable gene segmentscomprises the proximal repeat (with respect to the immunoglobulin κconstant region) of a human immunoglobulin κ light chain locus. In someembodiments, the one or more human immunoglobulin κ light chain variablegene segments comprises at least 40Vκ gene segments, at least five Jκgene segments or a combination thereof of a human immunoglobulin κ lightchain locus.

In particular embodiments, the genetically modified non-human animalsfurther comprise a nucleotide sequence encoding an unrearranged humanimmunoglobulin light chain (V_(L)) gene segment and an unrearrangedhuman immunoglobulin light chain (J_(L)) gene segment. In someembodiments, the nucleotide sequence encoding the unrearranged lightchain V gene segment and the unrearranged light chain J gene segment isoperably linked to an immunoglobulin light chain constant region genesequence. In other embodiments, the nucleotide sequence encoding theunrearranged light chain V gene segment and the unrearranged light chainJ gene segment is operably linked to an immunoglobulin heavy chainconstant region gene sequence. In some embodiments, the unrearrangedhuman immunoglobulin light chain V (V_(L)) gene segment and theunrearranged human immunoglobulin J (J_(L)) gene segment are operablylinked, at an endogenous rodent locus, to a rodent immunoglobulin lightchain constant region gene; e.g., a κ or λ light chain constant regiongene.

In various embodiments, the unrearranged human variable region genesegments (e.g., human Vκ gene segments) are capable of rearranging andencoding human variable domains of an antibody. In some embodiments, thenon-human animal does not comprise an endogenous V_(L) gene segment. Insome embodiments, the human Vκ gene segments expressed by the non-humananimals are selected from the group consisting of Vκ1-5, Vκ1-6, Vκ1-8,Vκ1-9, Vκ1-12, Vκ1-13, Vκ1-16, Vκ1-17, Vκ1-22, Vκ1-27, Vκ1-32, Vκ1-33,Vκ1-35, Vκ1-37, Vκ1-39, Vκ1D-8, Vκ1 D-12, Vκ1 D-13, Vκ1 D-16, Vκ1 D-17,Vκ1 D-22, Vκ1 D-27, Vκ1 D-32, Vκ1 D-33, Vκ1 D-35, Vκ1D-37, Vκ1 D-39, Vκ1D-42, Vκ1D-43, Vκ1-NL1, Vκ2-4, Vκ2-10, Vκ2-14, Vκ2-18, Vκ2-19, Vκ2-23,Vκ2-24, Vκ2-26, Vκ2-28, Vκ2-29, Vκ2-30, Vκ2-36, Vκ2-38, Vκ2-40, Vκ2D-10,Vκ2D-14, Vκ2D-18, Vκ2D-19, Vκ2D-23, Vκ2D-24, Vκ2D-26, Vκ2D-28, Vκ2D-29,Vκ2D-30, Vκ2D-36, Vκ2D-38, Vκ2D-40, Vκ3-7, Vκ3-11, Vκ3-15, Vκ3-20,Vκ3-25, Vκ3-31, Vκ3-34, Vκ3D-7, Vκ3D-7, Vκ3D-11, Vκ3D-15, Vκ3D-15,Vκ3D-20, Vκ3D-25, Vκ3D-31, Vκ3D-34, V_(κ)3-NL1, Vκ3-NL2, Vκ3-N L3,Vκ3-NL4, Vκ3-NL5, Vκ4-1, Vκ5-2, Vκ6-21, Vκ6D-21, Vκ6D-41, and Vκ7-3. Insome embodiments, the genetically modified non-human animals describedherein express all functional human Vκ genes. In some embodiments, thehuman Vκ gene segments comprise Vκ4-1, Vκ5-2, Vκ7-3, Vκ2-4, Vκ1-5, andVκ1-6. In some embodiments, the Vκ gene segments comprise Vκ3-7, Vκ1-8,Vκ1-9, Vκ2-10, Vκ3-11, Vκ1-12, Vκ1-13, Vκ2-14, Vκ3-15 and Vκ1-16. Insome embodiments, the human Vκ gene segments comprise Vκ1-17, Vκ2-18,Vκ2-19, Vκ3-20, Vκ6-21, Vκ1-22, Vκ1-23, Vκ2-24, Vκ3-25, Vκ2-26, Vκ1-27,Vκ2-28, Vκ2-29, and Vκ2-30. In some embodiments, the human Vκ genesegments comprise Vκ3-31, Vκ1-32, Vκ1-33, Vκ3-34, Vκ1-35, Vκ2-36,Vκ1-37, Vκ2-38, Vκ1-39, and Vκ2-40. In various embodiments, thenon-human animal comprises five human Jκ gene segments, e.g., Jκ1, Jκ2,Jκ3, Jκ4, and Jκ5 gene segments. In specific embodiments, the Vκ genesegments comprise contiguous human immunoglobulin κ gene segmentsspanning the human immunoglobulin κ light chain locus from Vκ4-1 throughVκ2-40, and the Jκ gene segments comprise contiguous gene segmentsspanning the human immunoglobulin κ light chain locus from Jκ1 throughJκ5. In some embodiments, the immunoglobulin light chain locus comprisestwo human V_(L) gene segments, Vκ1-39 and Vκ3-20. In some embodiments,one or more (e.g., 2, 3, 4, or 5) human V_(L) gene segments and two ormore human J_(L) gene segments are present at an endogenous light chainlocus, e.g., at an endogenous kappa light chain locus. In someembodiments, the genetically modified non-human animal is a mouse thatcomprises a functional λ light chain locus. In other embodiments, themouse comprises a non-functional λ light chain locus. In someembodiments, the one or more human V_(H), one or more human D_(H), andone or more human J_(H) gene segments are operably linked to a mouse ora rat heavy chain constant region sequence (i.e., the one or more humanV_(L) gene segments and two or more human J_(L) gene segments arepresent at an endogenous heavy chain locus).

In some embodiments, a genetically modified non-human animal (e.g.,mouse or rat) as described herein expresses a rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence (i.e.,produces an antigen-binding protein comprising a rearranged heavy chainvariable domain) and one or more, two or more, three or more, four ormore, five or more, etc. light chain variable domains encoded by Vκgenes selected from the group consisting of Vκ1-5, Vκ1-6, Vκ1-8, Vκ1-9,Vκ1-12, Vκ1-13, Vκ1-16, Vκ1-17, Vκ1-22, Vκ1-27, Vκ1-32, Vκ1-33, Vκ1-35,Vκ1-37, Vκ1-39, Vκ1D-8, Vκ1 D-12, Vκ1 D-13, Vκ1 D-16, Vκ1 D-17, Vκ1D-22, Vκ1 D-27, Vκ1 D-32, Vκ1 D-33, Vκ1 D-35, Vκ1 D-37, Vκ1 D-39, Vκ1D-42, Vκ1 D-43, Vκ1-NL1, Vκ2-4, Vκ2-10, Vκ2-14, Vκ2-18, Vκ2-19, Vκ2-23,Vκ2-24, Vκ2-26, Vκ2-28, Vκ2-29, Vκ2-30, Vκ2-36, Vκ2-38, Vκ2-40, Vκ2D-10,Vκ2D-14, Vκ2D-18, Vκ2D-19, Vκ2D-23, Vκ2D-24, Vκ2D-26, Vκ2D- 28, Vκ2D-29,Vκ2D-30, Vκ2D-36, Vκ2D-38, Vκ2D-40, Vκ3-7, Vκ3-11, Vκ3-15, Vκ3-20,Vκ3-25, Vκ3-31, Vκ3-34, Vκ3D-7, Vκ3D-7, Vκ3D-11, Vκ3D-15, Vκ3D-15,Vκ3D-20, Vκ3D-25, Vκ3D-31, Vκ3D-34, Vκ3-NL1, Vκ3-N L2, Vκ3-NL3, Vκ3-NL4,Vκ3-NL5, Vκ4-1, Vκ5-2, Vκ6-21, Vκ6D-21, Vκ6D-41, and Vκ7-3.

In various embodiments, at least one of the light chain variable regiongene segments (e.g., human light chain variable region gene segments)encode one or more histidine codons that are not encoded by acorresponding human germline light chain variable gene segment. In someembodiments, the light chain variable domain as described hereinexhibits a decrease in dissociative half-life (t_(1/2)) at an acidic pHas compared to neutral pH of at least about 2-fold, at least about3-fold, at least about 4-fold, at least about 5-fold, at least about10-fold, at least about 15-fold, at least about 20-fold, at least about25-fold, or at least about 30-fold. In some embodiments, the decrease int₁₁₂ at an acidic pH as compared to a neutral pH is about 30 fold ormore. In some embodiments, at least one of the V_(L) gene segmentscomprises a substitution of at least one non-histidine codon encoded bythe corresponding human germline V_(L) segment sequence with a histidinecodon. In some embodiments, the substitution is of one, two, three, orfour codons (e.g., three or four codons). In some embodiments, thesubstitution is in the CDR3 codon(s). In some embodiments, the humanV_(L) gene segments are human Vκ1-39 and Vκ3-20 gene segments, and eachof the human Vκ1-39 and Vκ3-20 gene segments comprises a substitution ofat least one non-histidine codon encoded by a corresponding humangermline V_(L) gene segment with the histidine codon. In someembodiments, each of the human V_(κ)1-39 and V_(κ)3-20 gene segmentscomprises a substitution of three or four histidine codons. In someembodiments, the three or four substitutions are in the CDR3 region. Insome embodiments, the substitution is of three non-histidine codons ofthe human Vκ1-39 gene segment, wherein the substitution is designed toexpress histidines at positions 106, 108, and 111. In some embodiments,the substitution is of four non-histidine codons of the human Vκ1-39gene segment, and the substitution is designed to express histidines atpositions 105, 106, 108, and 111 (see, e.g., US 2013/0247234A1 and WO2013/138680, incorporated by reference herein). In some embodiments, thesubstitution is of three non-histidine codons of the human Vκ3-20 genesegment, and the substitution is designed to express histidines atpositions 105, 106, and 109. In yet additional embodiments, thesubstitution is of four non-histidine codons of the human Vκ3-20 genesegment, and the substitution is designed to express histidines atpositions 105, 106, 107, and 109. In some embodiments, theimmunoglobulin light chain locus comprises one or more but less than thewild type number of human V_(L) gene segments and one or more, e.g., twoor more, human J_(L) gene segments, wherein each of the human V_(L) genesegments comprises at least one histidine codon that is not encoded bythe corresponding human germline V_(L) gene segment. In variousembodiments, the non-human animal comprising the genetically modifiedimmunoglobulin loci as described herein, upon stimulation by an antigenof interest, expresses an antigen-binding protein comprising an aminoacid sequence derived from the human V_(L) gene segments, wherein theantigen-binding protein retains at least one histidine residue at anamino acid position encoded by the at least one histidine codonintroduced into the human V_(L) gene segment. In some embodiments, theanimal expresses a population of antigen-binding proteins in response toan antigen, wherein all antigen-binding proteins in the populationcomprise (a) immunoglobulin light chain variable domains derived from arearrangement of the human V_(L) gene segments and the J_(L) genesegments, wherein at least one of the human V_(L) gene segments encodesone or more histidine codons that are not encoded by the correspondinghuman germline V_(L) gene segment, and (b) immunoglobulin heavy chainscomprising human heavy chain variable domains encoded by the rearrangedhuman immunoglobulin heavy chain variable region nucleotide sequence.

Various embodiments encompass light chain constant region sequences. Insome embodiments, for example, a first nucleotide sequence that encodesthe rearranged heavy chain variable domain (i.e., where the firstnucleotide sequence is a rearranged human immunoglobulin heavy chainvariable region nucleotide sequence) is operably linked to a heavy chainconstant region gene sequence, and a second nucleotide sequence thatencodes the human light chain variable domain (i.e., where the secondnucleotide sequence is an unrearranged human immunoglobulin light chainvariable nucleotide sequence) is operably linked to a light chainconstant region gene sequence. In some embodiments, a first nucleotidesequence that encodes the rearranged heavy chain variable domain (i.e.,where the first nucleotide sequence is a rearranged human immunoglobulinheavy chain variable region nucleotide sequence) is operably linked to alight chain constant region gene sequence, and a second nucleotidesequence that encodes the human light chain variable domain (i.e., wherethe second nucleotide sequence is an unrearranged human immunoglobulinlight chain variable nucleotide sequence) is operably linked to a heavychain constant region gene sequence. In various embodiments, the lightchain constant region sequence operably linked to the rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence is ahuman κ light chain constant region sequence. In some embodiments, thelight chain constant region sequence operably linked to the rearrangedheavy chain variable domain is a mouse κ light chain constant regionsequence. In some embodiments, the light chain constant region sequenceoperably linked to the rearranged heavy chain variable domain is a rat κlight chain constant region sequence. In some embodiments, the lightchain constant region sequence operably linked to the rearranged heavychain variable domain is a human λ light chain constant region sequence.In some embodiments, the light chain constant region sequence operablylinked to the rearranged heavy chain variable domain is a mouse λ lightchain constant region sequence. In some embodiments, the light chainconstant region sequence operably linked to the rearranged heavy chainvariable domain is a rat λ light chain constant region sequence.

In various aspects, non-human animals are provided comprising agenetically modified immunoglobulin locus that encodes a rearrangedheavy chain variable domain (i.e., where an immunoglobulin locuscomprises a rearranged human immunoglobulin heavy chain variable regionnucleotide sequence), wherein the rearranged heavy chain variable domaincomprises a heavy chain variable (V_(H)) sequence that is operablylinked, via a spacer, to a heavy chain J segment (J_(H)) sequence,wherein the spacer comprises at least one amino acid residue. In someembodiments, the rearranged human immunoglobulin heavy chain variableregion nucleotide sequence is operably linked to a non-human heavy chainconstant region gene sequence. In some embodiments, the non-human heavychain constant region gene sequence is a mouse or a rat constant regiongene sequence. In some embodiments, the rearranged human immunoglobulinheavy chain variable region nucleotide sequence is operably linked to ahuman heavy chain constant region gene sequence. In some embodiments,the heavy chain constant region comprises a sequence selected from aC_(H)1, a hinge, a C_(H)2, a C_(H)3, and a combination thereof. In someembodiments, the rearranged human immunoglobulin heavy chain variableregion nucleotide sequence is operably linked to a non-human light chainconstant region gene sequence. In some embodiments, the non-human lightchain constant region gene sequence is a mouse or a rat constant regiongene sequence. In some embodiments, the rearranged human immunoglobulinheavy chain variable region nucleotide sequence is operably linked to ahuman light chain constant region gene sequence. In some embodiments,the spacer is a single amino acid residue. In some embodiments, thespacers are two amino acid residues. In some embodiments, the spacersare three amino acid residues. In some embodiments, the spacers are fouramino acid residues. In some embodiments, the spacers are five aminoacid residues. In some embodiments, the spacers are six amino acidresidues.

In another aspect, genetically modified non-human animals and methodsfor making said animals are provided in which the animals comprise afunctional universal light chain (“ULC”) immunoglobulin locus. In someembodiments, such animals further comprise a rearranged heavy chainvariable domain locus (i.e., a heavy chain variable domainimmunoglobulin locus comprising a rearranged human immunoglobulin heavychain variable region nucleotide sequence). A ULC is a common lightchain that can be used in a bispecific format that contains a function,e.g., a modification that affects FcRn binding to improve a half-life,e.g., a bispecific that comprises a heavy chain that binds an antigenand a light chain that binds FcRn. For example, the genetically modifiedmice as described herein are immunized with FcRN, to obtain antibodiesthat bind FcRN solely through the light chains. These light chainsproduced by the genetically modified non-human animal are used as ULCsthat assist the bispecific antibody to associate with an FcRn, therebyhelping to increase half-life. The remainder of the antibody (e.g.,either a second, different light chain, or a heavy chain that binds anantigen different than FcRn) is selected to perform a second function. AULC as used in the embodiments described herein can also be used togenerate antibody variable chain sequences whose diversity resultsprimarily from the processes of somatic mutation (e.g., hypermutation),thereby elucidating antibody variable chain sequences whoseantigen-binding capacity benefits from post-genomic events.

Various aspects include genetically modified non-human animalscomprising in their genomes a rearranged human heavy chain variableregion nucleic acid sequence, and further comprising in their genomes anucleic acid encoding a light chain variable domain as described hereincloned onto a constant region nucleic acid sequence selected from akappa constant region, a lambda constant region, a heavy chain constantregion (e.g., selected from the group consisting of a CH1, a hinge, aCH2, a CH3, and a combination thereof). In some embodiments, the lightchain variable region nucleic acid sequence is cloned onto a first humanheavy chain constant region nucleic acid sequence, and a second lightchain variable domain is cloned onto a second human heavy chain constantregion nucleic acid sequence; wherein the first and the second humanheavy chain constant region nucleic acid sequence are the same, thefirst light chain variable domain specifically binds a first antigen,and the second light chain variable domain specifically binds a secondantigen. In these embodiments, a dimer of two polypeptides is formed,wherein each of the light chain variable domains fused to the heavychain constant region exhibit distinct antigen-binding specificity.

In another aspect, a genetically modified non-human animal (e.g., mouse)is provided that is capable of producing a light chain that binds areceptor or other moiety that traverses the blood-brain barrier, e.g.,the transferrin receptor. Previous studies have shown that low affinityantibodies directed against the transferrin receptor will traverse theblood-brain barrier and be released due to low affinity. Thus, in someembodiments, the genetically modified animals (e.g., mice) describedherein are used to make a low affinity antibody to a moiety that iscapable of traversing the blood-brain barrier (e.g., a transferrinreceptor), wherein the low affinity antibody is bispecific and comprisesa second binding specificity to a desired target (i.e., the antibodybinds the traversing moiety, and also binds a different target than thetraversing moiety).

Methods of making and using the genetically modified non-human animalsdescribed herein are provided. Methods are provided for placing arearranged human heavy chain variable region nucleic acid sequence inoperable linkage with an immunoglobulin heavy or light chain constantregion nucleic acid sequence in the genome of a non-human animal. Invarious embodiments, the constant region nucleic acid sequence is humanor non-human, and the non-human animal is a rodent. In variousembodiments, the methods comprise making a non-human animal that furthercomprises an immunoglobulin light chain locus comprising one or more butless than the wild type number of human light chain variable region genesegments, e.g., two human V_(κ) gene segments and one or more humanJ_(κ) gene segments, operably linked to a human or non-human light chainconstant region nucleic acid sequence. In various aspects, the methodscomprise placing the aforementioned sequences in the germline of anon-human animal, e.g., a rodent, employing, e.g., transgenic technologyincluding, e.g., employing modified pluripotent or totipotent donorcells (e.g., ES cells or iPS cells) with host embryos, germ cells (e.g.,oocytes), etc. Thus, embodiments include a non-human immunoglobulinheavy chain locus in a genome of a non-human germ cell comprising arearranged human immunoglobulin heavy chain variable region nucleotidesequence operably linked to a heavy chain constant region gene sequence,wherein the constant region gene sequence comprises a non-humansequence, a human sequence, or a combination thereof. In someembodiments, the rearranged human immunoglobulin heavy chain variableregion nucleotide sequence is operably linked to an endogenous non-humanimmunoglobulin constant region gene sequence. In some embodiments, theendogenous non-human immunoglobulin constant region gene sequence is amouse or a rat heavy chain constant region gene sequence.

In various aspects, a method of making a non-human animal that comprisesa genetically modified immunoglobulin locus is provided, wherein themethod comprises: (a) modifying a genome of a non-human animal to deleteor render non-functional endogenous functional immunoglobulin heavychain V, D, and J gene segments; and (b) placing in the genome arearranged human immunoglobulin heavy chain variable region nucleotidesequence. In one such aspect, a method is provided for making anon-human animal that expresses a single immunoglobulin heavy chain froma rearranged heavy chain gene sequence in the germline of the non-humananimal, the method comprising a step of genetically modifying anon-human animal such that its entire antibody-expressing mature B cellpopulation expresses a heavy chain derived from (i) a single V_(H) genesegment; (ii) an amino acid spacer of one, two, three, four, five, orsix amino acids; and (iii) a single J_(H) gene segment. In some aspects,the method comprises inactivating or replacing an endogenous heavy chainimmunoglobulin variable locus with a single rearranged heavy chain geneas described herein.

In another aspect, methods of making a non-human animal that comprises agenetically modified immunoglobulin heavy chain locus are provided, suchmethods comprising: (a) modifying a genome of a non-human animal todelete or render non-functional endogenous functional immunoglobulinheavy chain V, D, and J gene segments; and (b) placing in the genome arearranged human immunoglobulin heavy chain variable region nucleotidesequence. In some embodiments, substantially all endogenous functionalV_(H), D, and J_(H) gene segments are deleted from the immunoglobulinheavy chain locus of the non-human animal or rendered non-functional(e.g., via insertion of a nucleotide sequence (e.g., an exogenousnucleotide sequence in the immunoglobulin locus or via non-functionalrearrangement, or inversion of, endogenous V_(H), D, J_(H) segments). Insome embodiments, the method comprises inserting a rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence (i.e., anucleotide sequence that encodes the rearranged heavy chain variabledomain) into an endogenous location (i.e., targeted to where thenucleotide sequence is located in a wild type non-human animal). In someembodiments, the rearranged human immunoglobulin heavy chain variableregion nucleotide sequence is present ectopically (e.g., at a locusdifferent from the endogenous immunoglobulin chain locus in its genome,or within its endogenous locus, e.g., within an immunoglobulin variablelocus, wherein the endogenous locus is placed or moved to a differentlocation in the genome). In some embodiments, e.g., about 80% or more,about 85% or more, about 90% or more, about 95% or more, about 96% ormore, about 97% or more, about 98% or more, or about 99% or more of allendogenous functional V, D, or J gene segments are deleted or renderednon-functional. In some embodiments, e.g., at least 95%, 96%, 97%, 98%,or 99% of endogenous functional heavy chain V, D, or J gene segments aredeleted or rendered non-functional.

In another aspect, methods are provided for making a non-human animalthat comprises a genetically modified immunoglobulin locus, comprising:(a) modifying a genome of a non-human animal to delete or rendernon-functional endogenous functional immunoglobulin light chain V and Jgene segments; and (b) placing in an endogenous immunoglobulin lightchain locus a rearranged human immunoglobulin heavy chain variableregion nucleotide sequence (i.e., a nucleotide sequence that encodes arearranged heavy chain variable domain), wherein the nucleotide sequenceis operably linked to a light chain constant region gene sequence. Insome embodiments, the genetically engineered immunoglobulin locus ispresent in the germline genome of the non-human animal. In someembodiments, the rearranged human immunoglobulin heavy chain variableregion nucleotide sequence is operably linked to a κ light chainconstant region gene sequence. In some embodiments, the rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence isoperably linked to a mouse or rat κ light chain constant region genesequence. In some embodiments, the rearranged human immunoglobulin heavychain variable region nucleotide sequence is operably linked to a humanκ light chain constant region gene sequence. In some embodiments, therearranged human immunoglobulin heavy chain variable region nucleotidesequence is operably linked to a λ light chain constant region genesequence. In some embodiments, rearranged human immunoglobulin heavychain variable region nucleotide sequence is operably linked to a mouseor rat λ light chain constant region gene sequence. In some embodiments,the rearranged human immunoglobulin heavy chain variable regionnucleotide sequence is operably linked to a human λ light chain constantregion gene sequence.

In another aspect, methods are provided for making a non-human animalthat comprises a genetically modified immunoglobulin locus, comprising:(a) modifying a genome of a non-human animal to delete or rendernon-functional: (i) endogenous functional immunoglobulin heavy chain V,D, and J gene segments, and (ii) endogenous functional immunoglobulinlight chain V and J gene segments; and (b) placing in the genome: (i) afirst nucleotide sequence that encodes a rearranged heavy chain variabledomain (i.e., where the first nucleotide sequence is a rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence), whereinthe first nucleotide sequence is operably linked to a light chainconstant region gene sequence, and (ii) a second nucleotide sequencethat encodes a human immunoglobulin light chain variable domain (i.e.,where the second nucleotide sequence is an unrearranged humanimmunoglobulin light chain variable region nucleotide sequence), whereinthe second nucleotide sequence is operably linked to a heavy chainconstant region gene sequence. In some embodiments, the geneticallyengineered immunoglobulin locus is present in the germline genome of thenon-human animal. In some embodiments, the first nucleotide sequencethat encodes the rearranged heavy chain variable domain is operablylinked to a κ light chain constant region gene sequence. In someembodiments, the first nucleotide sequence that encodes the rearrangedheavy chain variable domain is operably linked to a mouse or rat κ lightchain constant region gene sequence. In some embodiments, the firstnucleotide sequence that encodes the rearranged heavy chain variabledomain is operably linked to a human κ light chain constant region genesequence. In some embodiments, the first nucleotide sequence thatencodes the rearranged heavy chain variable domain is operably linked toa λ light chain constant region gene sequence. In some embodiments, thefirst nucleotide sequence that encodes the rearranged heavy chainvariable domain is operably linked to a mouse or rat λ light chainconstant region gene sequence. In some embodiments, the first nucleotidesequence that encodes the rearranged heavy chain variable domain isoperably linked to a human λ light chain constant region gene sequence.In some embodiments, the human immunoglobulin light chain variabledomain is a κ light chain variable domain. Thus, in some embodiments,the second nucleotide sequence is a human kappa light chain variableregion nucleotide sequence. In some embodiments, the humanimmunoglobulin light chain variable domain is a λ light chain variabledomain. Thus, in some embodiments, the second nucleotide sequence is ahuman lambda light chain variable region nucleotide sequence. In someembodiments, the heavy chain constant region gene sequence is anon-human immunoglobulin heavy chain constant region gene sequence. Insome embodiments, the non-human immunoglobulin heavy chain constantregion gene sequence is a mouse or a rat heavy chain constant regiongene sequence.

In another aspect, methods are provided for making a non-human animalthat comprises a genetically modified immunoglobulin locus, comprising:(a) modifying a genome of a non-human animal to delete or rendernon-functional: (i) endogenous functional immunoglobulin heavy chain V,D, and J gene segments, and (ii) endogenous functional immunoglobulinlight chain V and J gene segments; and (b) placing in the genome: (i) afirst nucleotide sequence that encodes a rearranged heavy chain variabledomain (i.e., where the first nucleotide sequence is a rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence), whereinthe first nucleotide sequence is operably linked to a heavy chainconstant region gene sequence, and (ii) a second nucleotide sequencethat encodes a human immunoglobulin light chain variable domain (i.e.,where the second nucleotide sequence is an unrearranged humanimmunoglobulin light chain variable region nucleotide sequence), whereinthe second nucleotide sequence is operably linked to a light chainconstant region gene sequence. In some embodiments, the light chainconstant region gene sequence is a κ light chain constant region genesequence. In some embodiments, the light chain constant region genesequence is a mouse or rat κ light chain constant region gene sequence.In some embodiments, the light chain constant region gene sequence is ahuman κ light chain constant region gene sequence. In some embodiments,the light chain constant region gene sequence is a λ light chainconstant region gene sequence. In some embodiments, the light chainconstant region gene sequence is a mouse or rat λ light chain constantregion gene sequence. In some embodiments, the light chain constantregion gene sequence is a human λ light chain constant region genesequence. In some embodiments, the human immunoglobulin light chainvariable domain is a κ light chain variable domain. In some embodiments,the human immunoglobulin light chain variable domain is a λ light chainvariable domain. In some embodiments, the heavy chain constant regiongene sequence is a non-human immunoglobulin heavy chain constant regiongene sequence. In some embodiments, the non-human immunoglobulin heavychain constant region gene sequence is a mouse or a rat heavy chainconstant region gene sequence.

In another aspect, a method of making a non-human animal that comprisesa genetically modified immunoglobulin heavy chain locus is providedcomprising: (a) modifying a genome of a non-human animal to delete orrender non-functional endogenous functional immunoglobulin heavy chainV, D, and J gene segments; and (b) placing in the genome a rearrangedhuman immunoglobulin heavy chain variable region nucleotide sequence,wherein the rearranged human immunoglobulin heavy chain variable regionnucleotide sequence comprises a heavy chain V gene segment (V_(H))sequence that is operably linked, via spacer, to a heavy chain J genesegment (J_(H)) sequence, wherein the spacer comprises at least oneamino acid residue. In some embodiments, the rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence isoperably linked to a non-human immunoglobulin heavy chain constantregion gene sequence. In some embodiments, the non-human immunoglobulinheavy chain constant region gene sequence is a mouse or ratimmunoglobulin heavy chain constant region gene sequence. In someembodiments, the rearranged human immunoglobulin heavy chain variableregion nucleotide sequence is operably linked to a non-humanimmunoglobulin light chain constant region gene sequence. In someembodiments, the non-human immunoglobulin light chain constant regiongene sequence is a mouse or rat immunoglobulin light chain constantregion gene sequence. In some embodiments, the rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence ispresent at an endogenous location (i.e., where the nucleotide sequenceis located in a wild-type non-human animal). In some embodiments therearranged human immunoglobulin heavy chain variable region nucleotidesequence is present ectopically (e.g., at a locus different from theendogenous immunoglobulin chain locus in its genome, or within itsendogenous locus, e.g., within an immunoglobulin variable locus, whereinthe endogenous locus is placed or moved to a different location in thegenome). In some embodiments, the spacers are a single amino acidresidue. In some embodiments, the spacers are two amino acid residues.In some embodiments, the spacers are three amino acid residues. In someembodiments, the spacers are four amino acid residues. In someembodiments, the spacers are five amino acid residues. In someembodiments, the spacers are six amino acid residues. In someembodiments, the nucleotide sequences encodes two copies, three copies,four copies, or more of the rearranged human immunoglobulin heavy chainvariable region nucleotide sequence operably linked to a heavy chainconstant domain gene sequence. In some embodiments, the nucleotidesequence encodes a plurality of copies of the rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence operablylinked to a heavy chain constant domain gene sequence.

Methods are provided for making a non-human animal, comprising: (a)modifying a genome of a non-human animal to delete or rendernon-functional (i) endogenous functional immunoglobulin heavy chainV_(H), D, and and/or J_(H) gene segments, and (ii) endogenous functionalimmunoglobulin light chain V and J gene segments; and (b) placing (i) arearranged heavy chain variable region nucleic acid sequence at a heavychain locus, wherein the rearranged heavy chain variable region nucleicacid sequence comprises a heavy chain V gene segment (V_(H)) sequencethat is operably linked, via spacer, to a heavy chain J gene segment(J_(H)) sequence, wherein the spacer comprises at least one amino acidresidue; and (ii) one or more but less than the wild type number ofhuman immunoglobulin light chain variable region gene segments (e.g.,two human V_(κ) gene segments and at least one human J_(κ) genesegments) operably linked to a human or non-human light chain constantregion nucleic acid sequence. In some embodiments, at least one of thelight chain variable region gene segments encodes one or more histidinecodons that are not encoded by a corresponding human germline lightchain variable gene segment.

In some aspects, a method for making a non-human animal comprising agenetically modified immunoglobulin locus is provided, comprising:

-   -   (a) modifying a genome of a non-human animal to delete or render        non-functional endogenous functional immunoglobulin light chain        V and J gene segments; and    -   (b) placing in the genome of the non-human animal a rearranged        human heavy chain variable region nucleotide sequence in        operable linkage to a light chain constant region nucleotide        sequence.        In various embodiments, the non-human animal is a rodent, e.g.,        a mouse, a rat, or a hamster. In some embodiments, the rodent is        a mouse. In some embodiments, the light chain constant region is        a rat or a mouse constant region, e.g., a rat or a mouse Cκ        constant region.

In another aspect, a method for making a non-human animal comprising agenetically modified immunoglobulin locus is provided, comprising:

-   -   (a) modifying a genome of a non-human animal to delete or render        non-functional:        -   (i) endogenous functional immunoglobulin heavy chain V, D,            and/or J gene segments, and        -   (ii) endogenous functional immunoglobulin light chain V and            J gene segments; and    -   (b) placing in the genome of the non-human animal:        -   (i) a first nucleotide sequence that encodes a rearranged            heavy chain variable domain (i.e., where the first            nucleotide sequence is a rearranged human immunoglobulin            heavy chain variable region nucleotide sequence), wherein            the first nucleotide sequence is operably linked to a light            chain constant region gene sequence, and        -   (ii) a second nucleotide sequence that encodes a human or            non-human light chain variable domain (i.e., where the            second nucleotide sequence is an unrearranged human            immunoglobulin light chain variable region nucleotide            sequence), wherein the second nucleotide sequence is            operably linked to a heavy chain constant region gene            sequence.

In various embodiments, the non-human animal is a rodent, e.g., a mouse,a rat, or a hamster. In some embodiments, the rodent is a mouse. In someembodiments, the light chain constant region is a rat or a mouseconstant region, e.g., a rat or a mouse Cκ constant region. In someembodiments, the second nucleotide sequence is operably linked to amouse or rat heavy chain constant region gene sequence selected from aC_(H)1, a hinge, a C_(H)2, a C_(H)3, and a combination thereof. In someembodiments, the second nucleotide sequence is operably linked to ahuman heavy chain constant region gene sequence selected from a C_(H)1,a hinge, a C_(H)2, a C_(H)3, and a combination thereof.

In another aspect, a method is provided for making a non-human animalthat comprises a genetically modified immunoglobulin locus, comprising:

-   -   (a) modifying a genome of a non-human animal to delete or render        non-functional:        -   (i) endogenous functional immunoglobulin heavy chain V, D,            and/or J gene segments, and        -   (ii) endogenous functional immunoglobulin light chain V and            J gene segments; and    -   (b) placing in the genome of the non-human animal:        -   (i) a first nucleotide sequence that encodes a rearranged            heavy chain variable domain (i.e., where the first            nucleotide sequence is a rearranged human immunoglobulin            heavy chain variable region nucleotide sequence), wherein            the first nucleotide sequence is operably linked to a heavy            chain constant region gene sequence; and        -   (ii) a second nucleotide sequence that encodes a light chain            variable domain (i.e., where the second nucleotide sequence            is an unrearranged human immunoglobulin light chain variable            region nucleotide sequence), wherein the second nucleotide            sequence is operably linked to a light chain constant region            gene sequence.

In another aspect, a method is provided for making a non-human animalthat comprises a genetically modified immunoglobulin locus, comprising:

-   -   (a) modifying a genome of a non-human animal to delete or render        non-functional:        -   (i) endogenous functional immunoglobulin heavy chain V, D,            and/or J gene segments, and        -   (ii) endogenous functional immunoglobulin light chain V and            J gene segments; and    -   (b) placing in the genome of the non-human animal:        -   (i) a first allele comprising:            -   (1) a first nucleotide sequence that encodes a                rearranged heavy chain variable domain (i.e., where the                first nucleotide sequence is a rearranged human                immunoglobulin heavy chain variable region nucleotide                sequence) operably linked to a heavy chain constant                region gene sequence, and            -   (2) a second nucleotide sequence that encodes a light                chain variable domain (i.e., where the second nucleotide                sequence is an unrearranged human immunoglobulin light                chain variable region nucleotide sequence) operably                linked to a light chain constant region gene sequence;                and        -   (ii) a second allele comprising            -   (1) a third nucleotide sequence that encodes a light                chain variable domain (i.e., where the third nucleotide                sequence is an unrearranged human immunoglobulin light                chain variable region nucleotide sequence) operably                linked to a heavy chain constant region gene sequence,                and            -   (2) a fourth nucleotide sequence that encodes the                rearranged heavy chain variable domain (i.e., where the                fourth nucleotide sequence is a rearranged human                immunoglobulin heavy chain variable region nucleotide                sequence) operably linked to a light chain constant                region gene sequence.

In another aspect, a method of making a non-human animal that comprisesa genetically modified immunoglobulin heavy chain locus is providedcomprising: (a) modifying a genome of a non-human animal to delete orrender non-functional endogenous functional immunoglobulin heavy chainV, D, and and/or J gene segments; and (b) placing in the genomerearranged human immunoglobulin heavy chain variable region nucleotidesequence, wherein the rearranged human immunoglobulin heavy chainvariable region nucleotide sequence comprises a heavy chain V genesegment (V_(H)) sequence that is operably linked, via spacer, to a heavychain J gene segment (J_(H)) sequence, wherein the spacer comprises atleast one amino acid residue.

In another aspect, a method for making a non-human animal comprising agenetically modified immunoglobulin locus is provided, comprising:

-   -   (a) modifying a genome of a non-human animal to delete or render        non-functional:        -   (i) endogenous functional immunoglobulin heavy chain V, D,            and/or J gene segments, and    -   (ii) endogenous functional immunoglobulin light chain V and J        gene segments; and    -   (b) placing in the genome of the non-human animal:        -   (i) a rearranged human immunoglobulin heavy chain variable            region nucleotide sequence in operable linkage to a heavy            chain constant region nucleotide sequence; and        -   (ii) one or more but less than the wild type number of human            immunoglobulin light chain variable region gene segments in            operable linkage to a light chain constant region nucleic            acid sequence.

In various embodiments, the non-human animal is a rodent, e.g., a mouse,a rat, or a hamster. In some embodiments, the rodent is a mouse. In someembodiments, the light chain constant region is a rat or a mouseconstant region, e.g., a rat or a mouse Cκ constant region. In someembodiments, the rearranged human heavy chain variable region nucleicacid sequence is operably linked to a mouse or rat heavy chain constantregion gene sequence selected from a C_(H)1, a hinge, a C_(H)2, aC_(H)3, and a combination thereof. In some embodiments, the rearrangedheavy chain variable region nucleic acid sequence is operably linked toa human heavy chain constant region gene sequence selected from aC_(H)1, a hinge, a C_(H)2, a C_(H)3, and a combination thereof.

In another aspect, a method for making a non-human animal comprising agenetically modified immunoglobulin locus is provided, comprising:

-   -   (a) modifying a genome of a non-human animal to delete or render        non-functional:        -   (i) endogenous functional immunoglobulin heavy chain V, D,            and/or J gene segments, and        -   (ii) endogenous functional immunoglobulin light chain V and            J gene segments; and    -   (b) placing in the genome of the non-human animal:        -   (i) a rearranged human immunoglobulin heavy chain variable            region nucleotide sequence in operable linkage to a light            chain constant region nucleotide sequence; and        -   (ii) one or more but less than the wild type number of human            immunoglobulin light chain variable V_(L) and J_(L) gene            segments in operable linkage to a heavy chain constant            region nucleic acid sequence.            In various embodiments, the non-human animal is a rodent,            e.g., a mouse, a rat, or a hamster. In some embodiments, the            rodent is a mouse. In some embodiments, the light chain            constant region is a rat or a mouse constant region, e.g., a            rat or a mouse Cκ constant region.

In another aspect, nucleic acid sequences encoding a rearranged heavychain variable domain (i.e., nucleotide sequences that are rearrangedhuman immunoglobulin heavy chain variable region nucleotide sequences;i.e., a pre-rearranged variable heavy chain VDJ nucleotide sequence) areprovided. In some embodiments, the nucleic acid sequence is derived froma human V, D, and J gene sequence or segment. In some embodiments, thenucleic acid sequence is derived from a human germline V segment, ahuman germline D segment, and a human germline J segment. In someembodiments, the human V_(H) segment corresponds to observed variants inthe human population. In various embodiments, the nucleic acid sequencecomprises a human V gene selected from the group consisting of V_(H)1-2,V_(H)1-3, V_(H)1-8, V_(H)1-18, V_(H)1-24, V_(H)1-45, V_(H)1-46,V_(H)1-58, V_(H)1-69, V_(H)2-5, V_(H)2-26, V_(H)2- 70, V_(H)3-7,V_(H)3-9, V_(H)3-11, V_(H)3-13, V_(H)3-15, V_(H)3-16, V_(H)3-20,V_(H)3-21, V_(H)3-23, V_(H)3-30, V_(H)3-30-3, V_(H) 3-30-5, V_(H)3-33,V_(H)3-35, V_(H)3-38, V_(H)3-43, V_(H)3-48, V_(H)3-49, V_(H)3-53,V_(H)3-64, V_(H)3-66, V_(H)3-72, V_(H)3-73, V_(H)3-74, V_(H)4-4,V_(H)4-28, V_(H)4-30-1, V_(H)4-30-2, V_(H)4-30-4, V_(H)4-31, V_(H)4-34,V_(H)4-39, V_(H)4-59, V_(H)4-61, V_(H)5-51, V_(H)6-1, V_(H)7-4-1,V_(H)7-81, and a polymorphic variant thereof. In some embodiments, thehuman V segment is V_(H)3-23 or polymorphic variant thereof. In variousembodiments, the nucleic acid sequence comprises a human D gene segmentselected from the group consisting of D1-1, D1-7, D1-14, D1-20, D1-26,D2-2, D2-8, D2-15, D2-21, D3-3, D3-9, D3-10, D3-16, D3-22, D4-4, D4-11,D4-17, D4-23, D5-12, D5-5, D5-18, D5-24, D6-6, D6-13, D6-19, D6-25,D7-27, and a polymorphic variant thereof. In some embodiments, thenucleic acid sequence comprises a human D segment that is notautoreactive (non-immunogenic) in the animal. In some embodiments, thenucleic acid sequence comprises a human D segment that is capable ofbeing expressed in a heavy chain variable sequence of a mature B cell ofa mouse. In some embodiments, the nucleic acid sequence furthercomprises a human or non-human animal heavy chain constant region genesequence selected from a C_(H)1, a hinge, a C_(H)2, a C_(H)3, and acombination thereof. In specific embodiments, the nucleic acid comprisesa constant region gene sequence comprising a C_(H)1, a hinge, a C_(H)2,and a C_(H)3. In various embodiments, the nucleic acid sequencecomprises a human J gene segment is selected from the group consistingof J_(H)1, J_(H)2, J_(H)3, 44, J_(H)S, J_(H)6, and a polymorphic variantthereof. In some embodiments, the rearranged human immunoglobulin heavychain variable region nucleotide sequence encodes the sequence of humanV_(H)3-23/GY/J_(H)4-4 (SEQ ID NO: 137). In some embodiments, the nucleicacid sequence encodes a rearranged heavy chain variable domaincomprising the sequence of human V_(H)3-23/X₁X₂/J (wherein X1 is anyamino acid, and X2 is any amino acid). In some embodiments, X₁ is Glyand X₂ is Tyr. In some embodiments, the nucleic acid sequence encodes arearranged heavy chain variable domain comprising the sequence of humanV_(H)3-23/X₁X₂/J_(H)4-4 (wherein X1 is any amino acid, and X₂ is anyamino acid). In some embodiments, X₂ is an amino acid comprising aphenyl group. In specific embodiments, X₂ is selected from Tyr and Phe.In some embodiments, the nucleic acid sequence further comprises a humanor non-human animal light chain constant region gene sequence.

In another aspect, a nucleic acid construct is provided comprising arearranged human immunoglobulin heavy chain variable region nucleotidesequence (i.e., a pre-rearranged heavy chain VDJ sequence) as describedherein. In some embodiments, the nucleic acid construct is designed insuch a way that the rearranged human immunoglobulin heavy chain variableregion nucleotide sequence is operably linked to a human or non-humananimal heavy chain constant region gene sequence. In some embodiments,the nucleic acid construct contains two copies, three copies, fourcopies, or more of the rearranged human immunoglobulin heavy chainvariable region nucleotide sequence operably linked to a heavy chainconstant region gene sequence. In some embodiments, the nucleic acidconstruct is a targeting vector. In some embodiments, the targetingvector comprises an Adam6a gene, an Adam6b gene, or both, in order toprevent fertility problems associated with the deletion of the Adam6a/6bgenes (see, for example, US 2012-0322108A1, incorporated by reference inits entirety). In some embodiments, the Adam6a and the Adam6b genes areplaced at 5′ upstream of the transcriptional unit of the universal heavychain sequence. In some embodiments, the targeting vector comprises aselection cassette flanked by recombination sites. In some embodiments,the targeting vector comprises one or more site-specific recombinationsites (e.g., a loxP or a FRT site).

In another aspect, methods are provided for obtaining a light chainvariable region (V_(L)) amino acid sequence capable of binding anantigen independently from a heavy chain variable region amino acidsequence, comprising: (a) immunizing a genetically modified non-humananimal as described herein (e.g., a genetically modified animalcomprising a rearranged human heavy chain variable region nucleic acidsequence in operable linkage to a heavy or light chain constant regionnucleic acid sequence) with an antigen of interest, wherein thenon-human animal mounts an immune response to the antigen; and (b)obtaining a rearranged light chain (VJ) nucleic acid sequence of a lightchain variable domain that specifically binds the antigen from a cell(e.g., mature B cell) of the genetically modified non-human animal. Invarious embodiments, the light chain variable regions produced by suchmethods are provided.

In some aspects, methods for obtaining a nucleic acid sequence thatencodes an immunoglobulin light chain variable region (V_(L)) domaincapable of binding an antigen independently from a heavy chain variableregion are provided, comprising: (a) immunizing a non-human animal withan antigen of interest or an immunogen thereof, wherein the non-humananimal comprises in its genome (i) a rearranged human immunoglobulinheavy chain variable region nucleic acid sequence operably linked to aheavy chain constant region nucleic acid sequence, (b) allowing thenon-human animal to mount an immune response, (c) isolating from theimmunized non-human animal a cell comprising a nucleic acid sequencethat encodes a light chain variable domain that can bind the antigen,and (d) obtaining from the cell a nucleic acid sequence that encodes thelight chain variable domain (V_(L) domain) that can bind the antigen. Insome embodiments, the heavy chain constant region gene sequence is amouse or rat heavy chain constant region gene sequence. In someembodiments, the heavy chain constant region gene sequence is a humanheavy chain constant region gene sequence. In some embodiments, therearranged heavy chain variable domain expressed by the geneticallymodified locus is not autoreactive, i.e., non-immunogenic to thenon-human animal. In some embodiments, the non-human animal furthercomprises in its genome two or more but less than the wild type numberof human immunoglobulin light chain variable region gene segments (V_(L)and J_(L)). In some embodiments, the human immunoglobulin light chainvariable region gene segments (V_(L) and J_(L)) are operably linked to alight chain constant region nucleic acid sequence. In some embodiments,the isolating step (c) is carried out via fluorescence-activated cellsorting (FACS) or flow cytometry. In some embodiments, the cellcomprising the nucleic acid sequence that encodes the light chainvariable domain that bind the antigen is a lymphocyte. In someembodiments, the lymphocyte comprises natural killer cells, T cells, orB cells. In some embodiments, the method further comprises a step of(c)′ fusing the lymphocyte with a cancer cell. In certain embodiments,the cancer cell is a myeloma cell.

Thus, in various aspects, methods are provided for obtaining a nucleicacid sequence that encodes an immunoglobulin light chain variable domain(V_(L)) capable of binding an antigen independently from a heavy chainvariable domain, comprising:

-   -   (a) immunizing a non-human animal with an antigen of interest or        an immunogen thereof, wherein the non-human animal comprises in        its genome (i) a rearranged human immunoglobulin heavy chain        variable region nucleic acid sequence operably linked to a heavy        chain constant region nucleic acid sequence; and (ii)        unrearranged human immunoglobulin light chain variable region        gene segments (V_(L) and J_(L)) operably linked to a light chain        constant region nucleic acid sequence;    -   (b) allowing the non-human animal to mount an immune response;    -   (c) isolating from the immunized non-human animal a cell        comprising a nucleic acid sequence that encodes a light chain        variable domain that can bind the antigen; and    -   (d) obtaining from the cell a nucleic acid sequence that encodes        the light chain variable domain (V_(L) domain) that can bind the        antigen.        In some embodiments, the isolating step (c) is carried out via        fluorescence-activated cell sorting (FACS) or flow cytometry. In        some embodiments, the cell comprsing the nucleic acid sequence        that encodes the light chain variable domain that bind the        antigen is a lymphocyte. In particular embodiments, the        lymphocyte comprises natural killer cells, T cells, or B cells.        In some embodiments, the methods further comprise a step of (c)′        fusing the lymphocyte with a cancer cell. In particular        embodiments, the cancer cell is a myeloma cell. In some        embodiments, the nucleic acid sequence of (d) is fused with a        nucleic acid sequence encoding an immunoglobulin constant region        nucleic acid sequence. In some embodiments, the light chain        constant region nucleic acid sequence is a human kappa sequence        or a human lambda sequence. In some embodiments, the light chain        constant region nucleic acid sequence is a mouse kappa sequence        or a mouse lambda sequence. In some embodiments, the light chain        constant region nucleic acid sequence is a rat kappa sequence or        a rat lambda sequence. In some embodiments, the heavy chain        constant region nucleic acid sequence is a human sequence        selected from a CH1, a hinge, a CH2, a CH3, and a combination        thereof. In some embodiments, the heavy chain constant region        nucleic acid sequence is a mouse or rat selected from a CH1, a        hinge, a CH2, a CH3, and a combination thereof. In some        embodiments, the nucleic acid sequence of (d) comprises one or        more histidine codon substitutions or insertions that are        derived from the unrearranged V_(L) gene segment in the genome        of the animal.

In various aspects, methods are provided for obtaining a nucleic acidsequence that encodes an immunoglobulin light chain variable domain(V_(L)) capable of binding an antigen independently from a heavy chainvariable domain, comprising:

-   -   (a) immunizing a non-human animal with an antigen of interest or        an immunogen thereof, wherein the non-human animal comprises in        its genome (i) a rearranged human immunoglobulin heavy chain        variable region nucleic acid sequence operably linked to a light        chain constant region nucleic acid sequence; and (ii)        unrearranged human immunoglobulin light chain variable region        gene segments (V_(L) and J_(L)) operably linked to a heavy chain        constant region nucleic acid sequence;    -   (b) allowing the non-human animal to mount an immune response;    -   (c) isolating from the immunized non-human animal a cell        comprising a nucleic acid sequence that encodes a light chain        variable domain that can bind the antigen; and    -   (d) obtaining from the cell a nucleic acid sequence that encodes        the light chain variable domain (V_(L) domain) that can bind the        antigen.        In some embodiments, the isolating step (c) is carried out via        fluorescence-activated cell sorting (FACS) or flow cytometry. In        some embodiments, the cell comprsing the nucleic acid sequence        that encodes the light chain variable domain that bind the        antigen is a lymphocyte. In particular embodiments, the        lymphocyte comprises natural killer cells, T cells, or B cells.        In some embodiments, the methods further comprise a step of (c)′        fusing the lymphocyte with a cancer cell. In particular        embodiments, the cancer cell is a myeloma cell. In some        embodiments, the nucleic acid sequence of (d) is fused with a        nucleic acid sequence encoding an immunoglobulin constant region        nucleic acid sequence. In some embodiments, the light chain        constant region nucleic acid sequence is a human kappa sequence        or a human lambda sequence. In some embodiments, the light chain        constant region nucleic acid sequence is a mouse kappa sequence        or a mouse lambda sequence. In some embodiments, the light chain        constant region nucleic acid sequence is a rat kappa sequence or        a rat lambda sequence. In some embodiments, the heavy chain        constant region nucleic acid sequence is a human sequence        selected from a CH1, a hinge, a CH2, a CH3, and a combination        thereof. In some embodiments, the heavy chain constant region        nucleic acid sequence is a mouse or rat selected from a CH1, a        hinge, a CH2, a CH3, and a combination thereof. In some        embodiments, the nucleic acid sequence of (d) comprises one or        more histidine codon substitutions or insertions that are        derived from the unrearranged V_(L) gene segment in the genome        of the animal.

In some aspects, methods are provided for obtaining a nucleic acidsequence that encodes an immunoglobulin light chain variable domain(V_(L)) capable of binding an antigen independently from a heavy chainvariable domain, comprising:

-   -   (a) immunizing a non-human animal with an antigen of interest or        an immunogen thereof, wherein the non-human animal comprises in        its genome (i) a rearranged human immunoglobulin heavy chain        variable region nucleic acid sequence operably linked to a heavy        chain constant region nucleic acid sequence; and (ii) two or        more but less than the wild type number of human immunoglobulin        light chain variable region gene segments (V_(L) and J_(L))        operably linked to a light chain constant region nucleic acid        sequence;    -   (b) allowing the non-human animal to mount an immune response;    -   (c) isolating from the immunized non-human animal a cell        comprising a nucleic acid sequence that encodes a light chain        variable domain that can bind the antigen; and    -   (d) obtaining from the cell a nucleic acid sequence that encodes        the light chain variable domain (V_(L) domain) that can bind the        antigen.        In some embodiments, the isolating step (c) is carried out via        fluorescence-activated cell sorting (FACS) or flow cytometry. In        some embodiments, the cell comprsing the nucleic acid sequence        that encodes the light chain variable domain that bind the        antigen is a lymphocyte. In particular embodiments, the        lymphocyte comprises natural killer cells, T cells, or B cells.        In some embodiments, the methods further comprise a step of (c)′        fusing the lymphocyte with a cancer cell. In particular        embodiments, the cancer cell is a myeloma cell. In some        embodiments, the nucleic acid sequence of (d) is fused with a        nucleic acid sequence encoding an immunoglobulin constant region        nucleic acid sequence. In some embodiments, the light chain        constant region nucleic acid sequence is a human kappa sequence        or a human lambda sequence. In some embodiments, the light chain        constant region nucleic acid sequence is a mouse kappa sequence        or a mouse lambda sequence. In some embodiments, the light chain        constant region nucleic acid sequence is a rat kappa sequence or        a rat lambda sequence. In some embodiments, the heavy chain        constant region nucleic acid sequence is a human sequence        selected from a CH1, a hinge, a CH2, a CH3, and a combination        thereof. In some embodiments, the heavy chain constant region        nucleic acid sequence is a mouse or rat selected from a CH1, a        hinge, a CH2, a CH3, and a combination thereof. In some        embodiments, the nucleic acid sequence of (d) comprises one or        more histidine codon substitutions or insertions that are        derived from the unrearranged V_(L) gene segment in the genome        of the animal.

In some aspects, methods are provided for obtaining a nucleic acidsequence that encodes an immunoglobulin light chain variable domain(V_(L)) capable of binding an antigen independently from a heavy chainvariable domain, comprising:

-   -   (a) immunizing a non-human animal containing a genetically        modified immunoglobulin locus as described herein with an        antigen of interest, wherein the non-human animal comprises in        its genome a rearranged human immunoglobulin heavy chain        variable region nucleic acid sequence operably linked to a heavy        chain constant region nucleic acid sequence;    -   (b) allowing the non-human animal to mount an immune response;    -   (c) harvesting a lymphocyte (e.g., a B cell) from the immunized        non-human animal;    -   (d) fusing the lymphocyte with a myeloma cell to form a        hybridoma cell; and    -   e) obtaining from the hybridoma cell a nucleic acid sequence        that encodes a light chain variable domain (V_(L) domain) that        can bind the antigen.

In another aspect, methods are provided for obtaining a nucleic acidsequence that encodes an immunoglobulin light chain variable domain(V_(L)) nucleic acid sequence of an immunoglobulin light chain capableof binding an antigen independently from a heavy chain variable region,comprising:

-   -   (a) immunizing a non-human animal containing a genetically        modified immunoglobulin locus as described herein with an        antigen of interest, wherein the non-human animal comprises in        its genome a rearranged human immunoglobulin heavy chain        variable region nucleic acid sequence operably linked to a heavy        chain constant region nucleic acid sequence;    -   (b) allowing the non-human animal to mount an immune response;    -   (c) identifying a lymphocyte (e.g., a B cell) from the immunized        non-human animal that expresses a V_(L) amino acid sequence that        binds the antigen independently from a heavy chain variable        region; and,    -   (d) cloning a nucleic acid sequence encoding the V_(L) amino        acid sequence of (c) from the lymphocyte of (c).

In another aspect, methods are provided for obtaining an immunoglobulinlight chain variable region (V_(L)) amino acid sequence capable ofbinding an antigen independently from a heavy chain variable region,comprising:

-   -   (a) immunizing a non-human animal containing a genetically        modified immunoglobulin locus as described herein with an        antigen of interest, wherein the non-human animal comprises in        its genome (i) a first nucleotide sequence that encodes a        rearranged heavy chain variable domain (i.e., where the first        nucleotide sequence is a rearranged human immunoglobulin heavy        chain variable region nucleotide sequence), wherein the first        nucleotide sequence is operably linked to a light chain constant        region gene sequence; and (ii) a second nucleotide sequence that        encodes a human or non-human light chain variable domain (i.e.,        where the second nucleotide sequence is an unrearranged human        immunoglobulin light chain variable nucleotide sequence),        wherein the second nucleotide sequence is operably linked to a        heavy chain constant region gene sequence;    -   (b) allowing the non-human animal to mount an immune response;    -   (c) harvesting a lymphocyte (e.g., a B cell) from the immunized        non-human animal;    -   (d) fusing the lymphocyte with a myeloma cell to form a        hybridoma cell; and    -   e) obtaining from the hybridoma cell a nucleic acid sequence        that encodes a light chain variable domain (V_(L) domain) that        can bind the antigen.

In another aspect, methods are provided for obtaining a nucleic acidsequence that encodes an immunoglobulin light chain variable domain(V_(L)) capable of binding an antigen independently from a heavy chainvariable domain, comprising:

-   -   (a) immunizing a non-human animal containing a genetically        modified immunoglobulin locus as described herein with an        antigen of interest, wherein the non-human animal comprises in        its genome (i) a first nucleotide sequence that encodes a        rearranged heavy chain variable domain (i.e., where the first        nucleotide sequence is a rearranged human immunoglobulin heavy        chain variable region nucleotide sequence), wherein the first        nucleotide sequence is operably linked to a light chain constant        region gene sequence; and (ii) a second nucleotide sequence that        encodes a human or non-human light chain variable domain (i.e.,        where the second nucleotide sequence is an unrearranged human        immunoglobulin light chain variable nucleotide sequence),        wherein the second nucleotide sequence is operably linked to a        heavy chain constant region gene sequence;    -   (b) allowing the non-human animal to mount an immune response;    -   (c) identifying a lymphocyte (e.g., a B cell) from the immunized        non-human animal that expresses a V_(L) amino acid sequence that        binds the antigen independently from a heavy chain variable        region; and,    -   (d) cloning a nucleic acid sequence encoding the VL amino acid        sequence of (c) from the lymphocyte of (c).

In another aspect, methods are provided for obtaining an immunoglobulinlight chain variable region (V_(L)) amino acid sequence capable ofbinding an antigen independently from a heavy chain variable region,comprising:

-   -   (a) immunizing a non-human animal containing a genetically        modified immunoglobulin locus as described herein with an        antigen of interest, wherein the non-human animal comprises in        its genome (i) a rearranged human immunoglobulin heavy chain        variable region nucleic acid sequence operably linked to a heavy        chain constant region nucleic acid sequence; and (ii) two or        more but less than the wild type number of human immunoglobulin        light chain variable region gene segments (V_(L) and J_(L));    -   (b) allowing the non-human animal to mount an immune response;    -   (c) harvesting a lymphocyte (e.g., a B cell) from the immunized        non-human animal;    -   (d) fusing the lymphocyte with a myeloma cell to form a        hybridoma cell; and    -   e) obtaining from the hybridoma cell a nucleic acid sequence        that encodes a light chain variable domain (V_(L) domain) that        can bind the antigen.

In another aspect, methods are provided for obtaining an immunoglobulinlight chain variable region (V_(L)) nucleic acid sequence of animmunoglobulin light chain capable of binding an antigen independentlyfrom a heavy chain variable region, comprising:

-   -   (a) immunizing a non-human animal containing a genetically        modified immunoglobulin locus as described herein with an        antigen of interest, wherein the non-human animal comprises in        its genome (i) a rearranged human immunoglobulin heavy chain        variable region nucleic acid sequence operably linked to a heavy        chain constant region nucleic acid sequence; and (ii) two or        more but less than the wild type number of human immunoglobulin        light chain variable region gene segments (V_(L) and J_(L));    -   (b) allowing the non-human animal to mount an immune response;    -   (c) identifying a lymphocyte (e.g., a B cell) from the immunized        non-human animal that expresses a V_(L) amino acid sequence that        binds the antigen independently from a heavy chain variable        region; and,    -   (d) cloning a nucleic acid sequence encoding the VL amino acid        sequence of (c) from the lymphocyte of (c).

In various embodiments, the light chain variable domain described hereinis an effector light chain variable domain. In some embodiments, theeffector light chain variable domain specifically binds FcRn in order toimprove a half-life of multispecific antibodies. For example, abispecific antibody comprises a heavy chain variable domain that bindsan antigen and a light chain variable domain that binds FcRn. In someembodiments, the genetically modified mice as described herein areimmunized with FcRN, to obtain antibodies that bind FcRN solely throughthe light chains. These light chains produced by the geneticallymodified non-human animal are used as universal or common light chainsthat assist the bispecific antibody to associate with an FcRn, therebyhelping to increase half-life. The remainder of the antibody (e.g.,either a second, different light chain, or a heavy chain that binds anantigen different than FcRn) is selected to perform a second function.

In additional aspects, a genetically modified immunoglobulin locusobtainable by any of the methods as described herein is provided. Invarious embodiments, the light chain variable regions produced by themethods as described herein and the nucleic acid sequence encoding suchlight chain variable regions are also provided.

In some aspects, an immunoglobulin locus in a germline genome of anon-human animal is provided comprising (1) a rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence that isoperably linked to a heavy chain constant region gene sequence, and (2)an unrearranged human immunoglobulin light chain variable regionnucleotide sequence that is operably linked to a light chain constantregion gene sequence. In some aspects, an immunoglobulin locus in agermline genome of a non-human animal is provided comprising (1) arearranged human immunoglobulin heavy chain variable region nucleotidesequence that is operably linked to a light chain constant region genesequence, and (2) an unrearranged human immunoglobulin light chainvariable region nucleotide sequence that is operably linked to a heavychain constant region gene sequence. In some aspects, an immunoglobulinlocus in a germline genome of a non-human animal is provided comprising(1) a rearranged human immunoglobulin heavy chain variable regionnucleotide sequence that is operably linked to a heavy chain constantregion gene sequence, and (2) a nucleotide sequence that encodes two ormore but less than the wild type number of human immunoglobulin lightchain variable region gene segments (V_(L) and J_(L)). In someembodiments, the light chain constant region gene sequence is a κ lightchain constant region gene sequence. In some embodiments, the lightchain constant region gene sequence is a λ light chain constant regiongene sequence. In some embodiments, the light chain constant region genesequence is a mouse or rat light chain constant region gene sequence. Insome embodiments, the light chain variable region nucleotide sequence isa κ light chain variable region gene sequence. In some embodiments, thelight chain variable region nucleotide sequence is a λ light chainvariable region gene sequence. In some embodiments, the light chainvariable region nucleotide sequence is a mouse or rat light chainvariable region gene sequence.

Additional aspects include antigen-binding proteins (e.g. antibodies)made by the genetically modified non-human animals described herein.Likewise, antigen-binding proteins (e.g., recombinant antibodies) withlight chain variable region (V_(L)) sequences derived from or producedby (i.e., expressed from the unrearranged human immunoglobulin lightchain variable region gene segments) the genetically modified non-humananimals described herein are also provided. In some embodiments, theantigen-binding proteins produced by the methods as described hereincomprise a heavy chain and a light chain, wherein the heavy chain doesnot interfere with the binding of the light chain to the antigen, and/orthe heavy chain does not bind the antigen in the absence of the lightchain. In some embodiments, the light chain variable domain binds anantigen of interest with a K_(D) that is no more than one order ofmagnitude higher in the absence of heavy chain than in the presence ofheavy chain (e.g., K_(D)˜10⁻¹⁰ in the presence of heavy chain orK_(D)˜10⁻⁹ in the absence of heavy chain). In some embodiments, theantigen-binding proteins as described herein include an immunoglobulinlight chain that can specifically bind an antigen of interest with anaffinity (K_(D)) lower than 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ or 10⁻¹⁰. In someembodiments, the immunoglobulin light chain produced by the methods arecapable of specifically binding an antigen of interest in the absence ofa heavy chain variable region with an affinity (K_(D)) lower than 10⁻⁶,10⁻⁷, 10⁻⁸, 10⁻⁹, or 10⁻¹⁰.

In various embodiments, the light chain variable domains generated asdescribed herein specifically bind a target molecule (“T”). A targetmolecule is any protein, polypeptide, or other macromolecule whoseactivity or extracellular concentration is desired to be attenuated,reduced or eliminated. In many instances, the target molecule to which alight chain variable region binds is a protein or polypeptide (i.e., a“target protein”); however, also provided are embodiments wherein thetarget molecule (“T”) is a carbohydrate, glycoprotein, lipid,lipoprotein, lipopolysaccharide, or other non-protein polymer ormolecule to which a light chain variable region binds. In variousembodiments, T can be a cell surface-expressed target protein or asoluble target protein. Target binding by the antigen-binding moleculemay take place in an extracellular or cell surface context. In certainembodiments, however, the antigen-binding molecule binds a targetmolecule inside the cell, for example within an intracellular componentsuch as the endoplasmic reticulum, Golgi, endosome, lysosome, etc.Examples of cell surface-expressed target molecules include cellsurface-expressed receptors, membrane-bound ligands, ion channels, andany other monomeric or multimeric polypeptide component with anextracellular portion that is attached to or associated with a cellmembrane. Non-limiting, exemplary cell surface-expressed targetmolecules that may be targeted by the multispecific antigen-bindingmolecules provided herein include, e.g., cytokine receptors (e.g.,receptors for IL-1, IL-4, IL-6, IL-13, IL-22, IL-25, IL-33, etc.), aswell as cell surface targets including other type 1 transmembranereceptors such as PRLR, G-protein coupled receptors such as GCGR, ionchannels such as Nav1.7, ASIC1 or ASIC2, non-receptor surface proteinssuch as MHC-I (e.g., HLA-B*27), etc. In embodiments in which T is a cellsurface-expressed target protein, the D1 component of the multispecificantigen-binding molecule can be, e.g., an antibody or antigen-bindingfragment of an antibody that specifically binds T, or a ligand orportion of a ligand that specifically interacts with the cellsurface-expressed target protein. For example, if T is IL-4R, the D1component can comprise or consist of IL-4 or a receptor-binding portionthereof. Examples of soluble target molecules include cytokines, growthfactors, and other ligands and signaling proteins. Non-limitingexemplary soluble target protein that may be targeted by themultispecific antigen-binding molecules provided herein include, e.g.,IL-1, IL-4, IL-6, IL-13, IL-22, IL-25, IL-33, SOST, DKK1, etc. Solubletargets molecules also include, e.g., non-human target molecules such asallergens (e.g., Fel D1, Betv1, CryJ1), pathogens (e.g., Candidaalbicans, S. aureus, etc.), and pathogenic molecules (e.g.,lipopolysaccharide (LPS), lipotechoic acid (LTA), Protein A., toxins,etc.). In embodiments in which T is a soluble target molecule, the D1component of the multispecific antigen-binding molecule can be, e.g., anantibody or antigen-binding fragment of an antibody that specificallybinds T, or a receptor or portion of a receptor that specificallyinteracts with the soluble target molecule. For example, if T is IL-4,the D1 component can comprise or consist of IL-4R or a ligand-bindingportion thereof. Target molecules also include tumor-associatedantigens.

In another aspect, antigen-binding proteins (e.g., bispecific ortrispecific antibodies) can be prepared utilizing antigen-specific lightchain variable domains derived from (i.e., with human light chainvariable region (V_(L)) sequences generated by) a non-human animalcomprising an immunoglobulin locus with a rearranged human heavy chainvariable region nucleic acid sequence (i.e., an animal comprising apredesigned, rearranged heavy chain VDJ sequence). Suchantigen-specific, reverse chimeric (e.g., human variable/mouse constant)light chains can be used to derive antigen-specific light chain variableregion sequences that can be cloned in-frame into an expression vectorwith a suitable human light chain constant region sequence. Anantigen-specific human heavy chain variable region(s) (specific for adifferent epitope on the same or different antigen than theantigen-specific light chain) from an animal comprising animmunoglobulin locus with a rearranged human heavy chain variable regionnucleic acid sequence (i.e., a mouse comprising a predesigned,rearranged heavy chain VDJ sequence), can be cloned in-frame into anexpression vector comprising human heavy chain constant region sequence,and the antigen-specific human light and heavy chains can beco-expressed in a suitable cell to obtain an antigen-binding protein(e.g., bispecific or trispecific human antibody). Alternatively, apreviously selected antigen-specific heavy chain, e.g., a heavy chainfrom an antibody that comprises a light chain derived from the samevariable region gene segment as the one used in the rearranged humanheavy chain variable region nucleic acid sequence may be cloned in-frameinto an expression vector comprising human heavy chain constant regionsequence, and the antigen-specific human light and heavy chains can beco-expressed in a suitable cell to obtain an antigen-binding protein(e.g., bispecific or trispecific human antibody). In some embodiments,the rearranged human immunoglobulin heavy chain variable regionnucleotide sequence is operably linked to a non-human heavy chainconstant region gene sequence (e.g., mouse or rat, kappa or lambda). Insome embodiments, the rearranged human immunoglobulin heavy chainvariable region nucleotide sequence is operably linked to a non-humanlight chain constant region gene sequence (e.g., mouse or rat, kappa orlambda). In some embodiments, the human light chain variable region(V_(L)) sequences are kappa gene sequences.

In another aspect, a method for making a multispecific antigen-bindingprotein is provided comprising:

-   -   (a) immunizing a first non-human animal containing a first        genetically modified immunoglobulin locus as described herein        with an antigen of interest, wherein the first non-human animal        comprises in its genome (i) a rearranged human immunoglobulin        heavy chain variable region nucleotide sequence operably linked        to a heavy chain constant region nucleic acid sequence;    -   (b) allowing the first non-human animal to mount an immune        response;    -   (c) harvesting a first lymphocyte (e.g., a B cell) from the        immunized first non-human animal, wherein the first lymphocyte        expresses affinity matured antibodies, wherein the affinity        matured antibodies comprise a human variable domain fused to a        non-human constant domain;    -   (d) identifying a nucleic acid sequence that encodes the human        light chain variable domain of the affinity matured antibodies;    -   (e) cloning the nucleic acid sequence of (d) in a first        expression construct in frame with a suitable human constant        region nucleic acid sequence (e.g., a human lambda or kappa        sequence) to form a first polypeptide gene;    -   (f) immunizing a second non-human animal containing a        genetically modified immunoglobulin locus as described herein        with a second antigen of interest, wherein the second non-human        animal comprises in its genome (i) unrearranged human V, D, and        J gene segments linked to a non-human heavy chain constant        region nucleic acid sequence; and (ii) a single rearranged human        light chain variable region sequence;    -   (g) allowing the second non-human animal to mount an immune        response;    -   (h) harvesting a second lymphocyte from the immunized second        non-human animal, wherein the second lymphocyte expresses        affinity matured antibodies, wherein the affinity matured        antibodies comprise a human heavy chain variable domain fused to        a non-human constant domain;    -   (i) identifying a nucleic acid sequence that encodes a human        heavy chain variable domain of the affinity matured antibodies        that specifically binds the second antigen;    -   (j) cloning the nucleic acid sequence of (i) in a second        expression construct in frame with a suitable human constant        region nucleic acid sequence (e.g., a human IgG1 constant        sequence) to form a second polypeptide gene; and    -   (k) introducing the first expression construct and the second        expression construct into a cell suitable for expressing the        first polypeptide gene and the second polypeptide gene so as to        form an antigen-binding protein comprising a dimer of the second        polypeptide, wherein each monomer of the second polypeptide is        associated with a monomer of the first polypeptide.        In various embodiments, the first expression construct and the        second expression construct are on separate vectors. In various        embodiments, the first expression construct and the second        expression construct are on the same vector. In various        embodiments, the first antigen and the second antigen are        different. In one embodiment, the first antigen and the second        antigen are the same. In various embodiments, the first antigen        is a cell surface receptor, and the second antigen is selected        from a soluble antigen and an antigen bound to a cell surface.        In specific embodiments, the first antigen is an Fc receptor        (e.g., an FcRN), the second antigen is a soluble protein, and        the antigen-binding protein comprises one or more histidine        substitutions and insertions derived from the V_(L) gene segment        in the genome of the non-human animal.

In another aspect, a method for making a multispecific antigen-bindingprotein is provided comprising:

-   -   (a) immunizing a first non-human animal containing a first        genetically modified immunoglobulin locus as described herein        with an antigen of interest, wherein the first non-human animal        comprises in its genome (i) a first nucleotide sequence that        encodes a rearranged heavy chain variable domain (i.e., where        the first nucleotide sequence is a rearranged human        immunoglobulin heavy chain variable region nucleotide sequence),        wherein the first nucleotide sequence is operably linked to a        light chain constant region gene sequence; and (ii) a second        nucleotide sequence that encodes a human or non-human light        chain variable domain (i.e., where the second nucleotide        sequence is an unrearranged human immunoglobulin light chain        variable nucleotide sequence), wherein the second nucleotide        sequence is operably linked to a heavy chain constant region        gene sequence;    -   (b) allowing the first non-human animal to mount an immune        response;    -   (c) harvesting a first lymphocyte (e.g., a B cell) from the        immunized first non-human animal, wherein the first lymphocyte        expresses affinity matured antibodies, wherein the affinity        matured antibodies comprise a human variable domain fused to a        non-human constant domain;    -   (d) identifying a nucleic acid sequence that encodes the human        light chain variable domain of the affinity matured antibodies;    -   (e) cloning the nucleic acid sequence of (d) in a first        expression construct in frame with a suitable human constant        region nucleic acid sequence (e.g., a human lambda or kappa        sequence) to form a first polypeptide gene;    -   (f) immunizing a second non-human animal containing a        genetically modified immunoglobulin locus as described herein        with a second antigen of interest, wherein the second non-human        animal comprises in its genome (i) unrearranged human V, D, and        J gene segments linked to a non-human heavy chain constant        region nucleic acid sequence; and (ii) a single rearranged human        light chain variable region sequence;    -   (g) allowing the second non-human animal to mount an immune        response;    -   (h) harvesting a second lymphocyte from the immunized second        non-human animal, wherein the second lymphocyte expresses        affinity matured antibodies, wherein the affinity matured        antibodies comprise a human heavy chain variable domain fused to        a non-human constant domain;    -   (i) identifying a nucleic acid sequence that encodes a human        heavy chain variable domain of the affinity matured antibodies        that specifically binds the second antigen;    -   (j) cloning the nucleic acid sequence of (i) in a second        expression construct in frame with a suitable human constant        region nucleic acid sequence (e.g., a human IgG1 constant        sequence) to form a second polypeptide gene; and    -   (k) introducing the first expression construct and the second        expression construct into a cell suitable for expressing the        first polypeptide gene and the second polypeptide gene so as to        form an antigen-binding protein comprising a dimer of the second        polypeptide, wherein each monomer of the second polypeptide is        associated with a monomer of the first polypeptide.        In various embodiments, the first expression construct and the        second expression construct are on separate vectors. In various        embodiments, the first expression construct and the second        expression construct are on the same vector. In some        embodiments, the first antigen and the second antigen are        different. In some embodiments, the first antigen and the second        antigen are the same. In various embodiments, the first antigen        is a cell surface receptor, and the second antigen is selected        from a soluble antigen and an antigen bound to a cell surface.        In specific embodiments, the first antigen is an Fc receptor        (e.g., an FcRN), the second antigen is a soluble protein, and        the antigen-binding protein comprises one or more histidine        substitutions and insertions derived from the V_(L) gene segment        in the genome of the non-human animal.

In another aspect, a method for making a multispecific antigen-bindingprotein is provided comprising:

-   -   (a) immunizing a first non-human animal containing a first        genetically modified immunoglobulin locus as described herein        with an antigen of interest, wherein the first non-human animal        comprises in its genome (i) a rearranged human immunoglobulin        heavy chain variable region nucleotide sequence operably linked        to a heavy chain constant region nucleic acid sequence; and (ii)        two or more but less than the wild type number of human        immunoglobulin light chain variable region gene segments (V_(L)        and J_(L));    -   (b) allowing the first non-human animal to mount an immune        response;    -   (c) harvesting a first lymphocyte (e.g., a B cell) from the        immunized first non-human animal, wherein the first lymphocyte        expresses affinity matured antibodies, wherein the affinity        matured antibodies comprise a human variable domain fused to a        non-human constant domain;    -   (d) identifying a nucleic acid sequence that encodes the human        light chain variable domain of the affinity matured antibodies;    -   (e) cloning the nucleic acid sequence of (d) in a first        expression construct in frame with a suitable human constant        region nucleic acid sequence (e.g., a human lambda or kappa        sequence) to form a first polypeptide gene;    -   (f) immunizing a second non-human animal containing a        genetically modified immunoglobulin locus as described herein        with a second antigen of interest, wherein the second non-human        animal comprises in its genome (i) unrearranged human V, D, and        J gene segments linked to a non-human heavy chain constant        region nucleic acid sequence; and (ii) a single rearranged human        light chain variable region sequence, wherein the single        rearranged human light chain variable region sequence is derived        from the same VL gene segment as the VL gene segment encoding        the light chain variable domain of step (c);    -   (g) allowing the second non-human animal to mount an immune        response;    -   (h) harvesting a second lymphocyte from the immunized second        non-human animal, wherein the second lymphocyte expresses        affinity matured antibodies, wherein the affinity matured        antibodies comprise a human heavy chain variable domain fused to        a non-human constant domain;    -   (i) identifying a nucleic acid sequence that encodes a human        heavy chain variable domain of the affinity matured antibodies        that specifically binds the second antigen;    -   (j) cloning the nucleic acid sequence of (i) in a second        expression construct in frame with a suitable human constant        region nucleic acid sequence (e.g., a human IgG1 constant        sequence) to form a second polypeptide gene; and    -   (k) introducing the first expression construct and the second        expression construct into a cell suitable for expressing the        first polypeptide gene and the second polypeptide gene so as to        form an antigen-binding protein comprising a dimer of the second        polypeptide, wherein each monomer of the second polypeptide is        associated with a monomer of the first polypeptide.        In various embodiments, the first expression construct and the        second expression construct are on separate vectors. In various        embodiments, the first expression construct and the second        expression construct are on the same vector. In various        embodiments, the first antigen and the second antigen are        different. In various embodiments, the first antigen and the        second antigen are the same. In various embodiments, the first        antigen is a cell surface receptor, and the second antigen is        selected from a soluble antigen and an antigen bound to a cell        surface. In various embodiments, the first antigen is an Fc        receptor (e.g., an FcRN), the second antigen is a soluble        protein, and the antigen-binding protein comprises one or more        histidine substitutions and insertions derived from the V_(L)        gene segment in the genome of the non-human animal.

In another aspect, a method for making a multispecific antigen-bindingprotein is provided comprising:

-   -   (a) immunizing a non-human animal containing a genetically        modified immunoglobulin locus as described herein with a first        antigen, wherein the non-human animal comprises in its        genome: (i) rearranged human immunoglobulin heavy chain variable        region nucleotide sequence operably linked to a heavy chain        constant region nucleic acid sequence; and (ii) one or more        human immunoglobulin V_(L) and J_(L) gene segments;    -   (b) allowing the non-human animal to mount an immune response;    -   (c) harvesting a lymphocyte (e.g., a B cell) from the immunized        non-human animal, wherein the lymphocyte expresses affinity        matured antibodies comprising a human immunoglobulin light chain        variable domain fused to a mouse immunoglobulin constant domain;    -   (d) identifying a first nucleic acid sequence that encodes the        human light chain variable domain of the affinity matured        antibodies;    -   (e) cloning the first nucleic acid sequence of (d) into a first        expression vector in frame with a human light chain constant        region nucleic acid sequence;    -   (f) introducing into a host cell: (i) the first expression        vector comprising the first nucleic acid sequence in frame with        the human light chain constant region nucleic acid sequence;        and (ii) a second expression vector comprising a second nucleic        acid sequence that encodes a first antigen-specific heavy chain        variable domain fused to a human heavy chain constant region;

(g) culturing the host cell to allow expression of multispecificantibodies; and

-   -   (h) isolating the multispecific antibodies, wherein the        multispecific antibodies comprise the first antigen-specific        heavy chain and the light chain variable domain, wherein the        heavy chain variable domain of the multispecific antibodies        exhibit an antigen binding specificity distinct from the light        chain variable domain.

In various embodiments, the multispecific antibodies are bispecificantibodies. In some embodiments, the multispecific antibodies aretrispecific antibodies, and step (f) further comprises introducing athird expression vector comprising a third nucleic acid sequence thatencodes a second antigen-specific heavy chain variable domain fused withthe human heavy chain constant region sequence.

In another aspect, a method for making a multispecific antigen-bindingprotein is provided comprising:

-   -   (a) immunizing a non-human animal containing a genetically        modified immunoglobulin locus as described herein with a first        antigen, wherein the non-human animal comprises in its        genome: (i) a rearranged heavy chain variable domain operably        linked to a heavy chain constant region nucleic acid sequence;        and (ii) one or more but less than the wild type number of human        immunoglobulin V_(L) and J_(L) gene segments;    -   (b) allowing the non-human animal to mount an immune response;    -   (c) harvesting a lymphocyte (e.g., a B cell) from the immunized        non-human animal, wherein the lymphocyte expresses affinity        matured antibodies comprising a human immunoglobulin light chain        variable domain fused to a mouse immunoglobulin constant domain;    -   (d) identifying a first nucleic acid sequence that encodes the        human light chain variable domain of the affinity matured        antibodies;    -   (e) cloning the first nucleic acid sequence of (d) into a first        expression vector in frame with a human light chain constant        region nucleic acid sequence;    -   (f) introducing into a host cell: (i) the first expression        vector comprising the first nucleic acid sequence in frame with        the human light chain constant region nucleic acid sequence;        and (ii) a second expression vector comprising a second nucleic        acid sequence that encodes a first antigen-specific heavy chain        variable domain fused to a human heavy chain constant region;    -   (g) culturing the host cell to allow expression of multispecific        antibodies; and    -   (h) isolating the multispecific antibodies, wherein the        multispecific antibodies comprise the first antigen-specific        heavy chain and the light chain variable domain, wherein the        heavy chain variable domain of the multispecific antibodies        exhibit an antigen binding specificity distinct from the light        chain variable domain.        In various embodiments, the multispecific antibodies are        bispecific antibodies. In some embodiments, the multispecific        antibodies are trispecific antibodies, and step (f) further        comprises introducing a third expression vector comprising a        third nucleic acid sequence that encodes a second        antigen-specific heavy chain variable domain fused with the        human heavy chain constant region sequence.

In another aspect, methods are provided for making an antigen-bindingprotein that comprises an immunoglobulin light chain variable domainthat can bind an antigen independently from a heavy chain variabledomain. Such methods comprise

-   -   (a) immunizing a genetically modified non-human animal with a        first antigen that comprises a first epitope or immunogenic        portion thereof, wherein the non-human animal comprises in its        genome:        -   (i) a rearranged human heavy chain variable region nucleic            acid sequence operably linked to a heavy chain constant            region nucleic acid sequence; and        -   (ii) unrearranged human immunoglobulin light chain variable            region gene segments (V_(L) and J_(L)) operably linked to an            immunoglobulin light chain constant region nucleic acid            sequence;    -   (b) allowing the non-human animal to mount an immune response to        the first epitope or immunogenic portion thereof;    -   (c) isolating from the non-human animal a cell comprising a        nucleic acid sequence that encodes a light chain variable domain        that specifically binds the first epitope or immunogenic portion        thereof;    -   (d) obtaining from the cell of (c) the nucleic acid sequence        that encodes the light chain variable domain that specifically        binds the first epitope or immunogenic portion thereof;    -   (e) employing the nucleic acid sequence of (d) in an expression        construct, fused to a human immunoglobulin constant region        nucleic acid sequence; and    -   (f) expressing the nucleic acid sequence of (d) in a production        cell line that expresses a human immunoglobulin heavy chain that        specifically binds a second antigen or epitope to form an        antigen-binding protein whose light chain is encoded by the        nucleic acid of (d) and that binds the first epitope or        immunogenic portion thereof independently from the heavy chain,        and whose heavy chain specifically binds the second antigen or        epitope.        In some embodiments at least one of the unrearranged human light        chain V_(L) or J_(L) gene segments encode one or more histidine        codons that are not encoded by a corresponding human germline        light chain variable gene segment. In some embodiments, the        first epitope is derived from a cell surface receptor. In some        embodiments, the cell surface receptor is an Fc receptor. In        particular embodiments, the Fc receptor is FcRn. In some        embodiments, the second antigen or epitope is derived from a        soluble antigen. In some embodiments, the second antigen or        epitope is derived from a cell surface receptor. In some        embodiments, the first antigen is an Fc receptor, the second        antigen is a soluble protein, and the antigen-binding protein        comprises one or more histidine substitutions and insertions        derived from the V_(L) gene segment in the genome of the        non-human animal.

In another aspect, methods are provided for making an antigen-bindingprotein that comprises an immunoglobulin light chain variable domainthat can bind an antigen independently from a heavy chain variabledomain. Such methods comprise

-   -   (a) immunizing a genetically modified non-human animal with a        first antigen that comprises a first epitope or immunogenic        portion thereof, wherein the non-human animal comprises in its        genome:        -   (i) a rearranged human heavy chain variable region nucleic            acid sequence operably linked to a light chain constant            region nucleic acid sequence; and        -   (ii) unrearranged human immunoglobulin light chain variable            region gene segments (V_(L) and J_(L)) operably linked to an            immunoglobulin heavy chain constant region nucleic acid            sequence;    -   (b) allowing the non-human animal to mount an immune response to        the first epitope or immunogenic portion thereof;    -   (c) isolating from the non-human animal a cell comprising a        nucleic acid sequence that encodes a light chain variable domain        that specifically binds the first epitope or immunogenic portion        thereof;    -   (d) obtaining from the cell of (c) the nucleic acid sequence        that encodes the light chain variable domain that specifically        binds the first epitope or immunogenic portion thereof;    -   (e) employing the nucleic acid sequence of (d) in an expression        construct, fused to a human immunoglobulin constant region        nucleic acid sequence; and    -   (f) expressing the nucleic acid sequence of (d) in a production        cell line that expresses a human immunoglobulin heavy chain that        specifically binds a second antigen or epitope to form an        antigen-binding protein whose light chain is encoded by the        nucleic acid of (d) and that binds the first epitope or        immunogenic portion thereof independently from the heavy chain,        and whose heavy chain specifically binds the second antigen or        epitope.        In some embodiments at least one of the unrearranged human light        chain V_(L) or J_(L) gene segments encode one or more histidine        codons that are not encoded by a corresponding human germline        light chain variable gene segment. In some embodiments, the        first epitope is derived from a cell surface receptor. In some        embodiments, the cell surface receptor is an Fc receptor. In        particular embodiments, the Fc receptor is FcRn. In some        embodiments, the second antigen or epitope is derived from a        soluble antigen. In some embodiments, the second antigen or        epitope is derived from a cell surface receptor. In some        embodiments, the first antigen is an Fc receptor, the second        antigen is a soluble protein, and the antigen-binding protein        comprises one or more histidine substitutions and insertions        derived from the V_(L) gene segment in the genome of the        non-human animal.

In another aspect, methods are provided for making an antigen-bindingprotein that comprises an immunoglobulin light chain variable domainthat can bind an antigen independently from a heavy chain variabledomain. Such methods comprise

-   -   (a) immunizing a genetically modified non-human animal with a        first antigen that comprises a first epitope or immunogenic        portion thereof, wherein the non-human animal comprises in its        genome:        -   (i) a rearranged human heavy chain variable region nucleic            acid sequence operably linked to a heavy chain constant            region nucleic acid sequence; and        -   (ii) two or more but less than the wild type number of human            immunoglobulin light chain variable region gene segments            (V_(L) and J_(L)) operably linked to an immunoglobulin light            chain constant region nucleic acid sequence;    -   (b) allowing the non-human animal to mount an immune response to        the first epitope or immunogenic portion thereof;    -   (c) isolating from the non-human animal a cell comprising a        nucleic acid sequence that encodes a light chain variable domain        that specifically binds the first epitope or immunogenic portion        thereof;    -   (d) obtaining from the cell of (c) the nucleic acid sequence        that encodes the light chain variable domain that specifically        binds the first epitope or immunogenic portion thereof;    -   (e) employing the nucleic acid sequence of (d) in an expression        construct, fused to a human immunoglobulin constant region        nucleic acid sequence; and    -   (f) expressing the nucleic acid sequence of (d) in a production        cell line that expresses a human immunoglobulin heavy chain that        specifically binds a second antigen or epitope to form an        antigen-binding protein whose light chain is encoded by the        nucleic acid of (d) and that binds the first epitope or        immunogenic portion thereof independently from the heavy chain,        and whose heavy chain specifically binds the second antigen or        epitope.        In some embodiments at least one of the human light chain V_(L)        or J_(L) gene segments encode one or more histidine codons that        are not encoded by a corresponding human germline light chain        variable gene segment. In some embodiments, the first epitope is        derived from a cell surface receptor. In some embodiments, the        cell surface receptor is an Fc receptor. In particular        embodiments, the Fc receptor is FcRn. In some embodiments, the        second antigen or epitope is derived from a soluble antigen. In        some embodiments, the second antigen or epitope is derived from        a cell surface receptor. In some embodiments, the first antigen        is an Fc receptor, the second antigen is a soluble protein, and        the antigen-binding protein comprises one or more histidine        substitutions and insertions derived from the V_(L) gene segment        in the genome of the non-human animal.

In another aspect, to allow for a facile separation of theantigen-binding proteins described herein, one of the heavy chains ismodified to omit a Protein A-binding determinant, resulting in adifferential Protein A-binding affinity of a homodimeric binding proteinfrom a heterodimeric binding protein. Compositions and methods thataddress this issue are described in U.S. Pat. No. 8,586,713, granted 19Nov. 2013, entitled “Readily Isolated Bispecific Antibodies with NativeImmunoglobulin Format,” hereby incorporated by reference. Once thespecie comprising heterodimeric heavy chain with an identical lightchain is selected, this bispecific antigen binding protein can bescreened to confirm the retention of its pH-dependent antigen bindingproperty.

In various aspects, a pluripotent cell, induced pluripotent, ortotipotent stem cells derived from a non-human animal comprising thevarious genomic modifications herein are provided. In some embodiments,the pluripotent or totipotent cell is derived from a non-human animal.In some embodiments, the non-human animal is a rodent, e.g., a mouse, arat, or a hamster. In some embodiments, the rodent is a mouse. Inspecific embodiments, the pluripotent cell is an embryonic stem (ES)cell. In some embodiments, the pluripotent cell comprises in its genome:(i) an immunoglobulin heavy chain locus that comprises a rearrangedhuman heavy chain variable region nucleic acid sequence operably linkedto a heavy chain constant region nucleic acid sequence; and (ii) animmunoglobulin light chain locus comprising one or more but less thanthe wild type number of human immunoglobulin light chain variable V_(L)and J_(L) gene segments, operably linked to a light chain constantregion nucleic acid sequence. In specific embodiments, the pluripotent,induced pluripotent, or totipotent stem cells are mouse or rat embryonicstem (ES) cells. In some embodiments, the pluripotent, inducedpluripotent, or totipotent stem cells have an XX karyotype or an XYkaryotype.

Cells that comprise a nucleus containing a genetic modification asdescribed herein are also provided, e.g., a modification introduced intoa cell by pronuclear injection. In another aspect, a hybridoma orquadroma is provided, derived from a cell of the non-human animal asdescribed herein. In some embodiments, the non-human animal is a rodent,such as a mouse, a rat, or a hamster.

In another aspect, a lymphocyte isolated from a genetically modifiednon-human animal as described herein is provided. In some embodiments,the lymphocyte is a B cell, wherein the B cell comprises animmunoglobulin genomic locus comprising a rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence operablylinked to a human or a non-human animal (e.g., mouse or rat) heavy chainor light chain constant region gene sequence. In some embodiments, the Bcell is capable of producing antibodies wherein the rearranged heavychain variable domain as described herein is operably linked to a heavychain or light chain constant domain.

In another aspect, a non-human animal embryo comprising a cell whosegenome comprises: (i) an immunoglobulin heavy chain locus comprising arearranged human heavy chain variable region nucleic acid sequenceoperably linked to a constant region nucleic acid sequence; and (ii) animmunoglobulin light chain locus comprising two or more but less thanthe wild type number of human immunoglobulin light chain variable regiongene segments, operably linked to a light chain constant region nucleicacid sequence.

In various embodiments, the genetically modified non-human animalsexpress an antibody repertoire (e.g., an IgG repertoire) that is derivedfrom the nucleotide sequence that encodes the rearranged heavy chainvariable domain, and a plurality of light chain V segments (and aplurality of light chain J segments). In some embodiments, thegenetically modified locus produces an antibody population thatcomprises an immunoglobulin light chain that is capable of specificallybinding an antigen of interest with an affinity (K_(D)) lower than 10⁻⁶,10⁻⁷, 10⁻⁸, 10⁻⁹ or 10⁻¹⁰. In some embodiments, the immunoglobulin lightchain expressed by the genetically modified locus is capable ofspecifically binding an antigen of interest in the absence of a heavychain variable region with an affinity (K_(D)) lower than 10⁻⁶, 10⁻⁷,10⁻⁸, 10⁻⁹, or 10⁻¹⁰.

In various embodiments, the genetic modifications described herein donot affect fertility of the non-human animal (see, for example, US2012-0322108A1, incorporated by reference in its entirety). In someembodiments, the heavy chain locus comprises an endogenous Adam6a gene,Adam6b gene, or both, and the genetic modification does not affect theexpression and/or function of the endogenous Adam6a gene, Adam6b gene,or both. In some embodiments, the genome of the genetically modifiednon-human animal comprises an ectopically located Adam6a gene, Adam6bgene, or both. In some embodiments, an Adam6a and/or Adam6b gene isplaced 5′ upstream of the transcriptional unit of the rearranged heavychain variable region nucleic acid sequence. In some embodiments, theAdam6a and/or the Adam6b gene is placed 3′ downstream of thetranscriptional unit of the rearranged heavy chain variable regionnucleic acid sequence.

In some embodiments, the genetically modified heavy chain locus does notcomprise an Intergenic Control Region 1 (IGCR1) nucleic acid sequence.In some embodiments, the genetically modified heavy chain locuscomprises an IGCR1 sequence downstream of the rearranged heavy chainvariable region nucleic acid sequence. In some embodiments, the IGCR1nucleic acid sequence is present between the rearranged heavy chainvariable region nucleic acid sequence and the most V-proximal D_(H) genesegment.

In some aspects, as noted earlier, the immunoglobulin light chain locusof the non-human animals described herein comprises a limited repertoireof light chain variable gene segments, e.g., (i) one, two or more butless than the wild type number of human V_(L) gene segments. In someembodiments, the non-human animal is a mouse; and the immunoglobulinlight chain variable domain is generated from a rearrangement of one oftwo human Vκ gene segments and one of 1, 2, 3, 4, or 5 human Jκ genesegments. In some embodiments, the mouse exhibits a ratio of (a) B cellsin the bone marrow that express an immunoglobulin having a λ light chainto (b) B cells in the bone marrow that express an immunoglobulin havinga κ light chain, of about 1 to about 15. In some embodiments, therearrangement includes a human Vκ1-39 gene segment. In some embodiments,the rearrangement includes a human Vκ3-20 gene segment. In someembodiments, the two human Vκ gene segments is at an endogenousimmunoglobulin Vκ locus, and, in some embodiments, the two human Vκ genesegments replace all or substantially all mouse immunoglobulin Vκ genesegments. In some embodiments, the two human Vκ gene segments are at anendogenous immunoglobulin Vκ locus, and, in some embodiments, the twohuman Vκ gene segments replace all or substantially all mouseimmunoglobulin Vκ and Jκ gene segments. In some embodiments, the twohuman Vκ gene segments are operably linked to two or more (e.g., 2, 3,4, 5) human Jκ gene segments. In some other embodiments, the light chainvariable domain of the mouse is generated through a rearrangement of ahuman Vκ1-39 gene segment or a human Vκ3-20 gene segment and one of twoor more (e.g., 2, 3, 4, or 5) human Jκ gene segments. In some suchembodiments, the ratio of immature B cells in the bone marrow thatexpress an immunoglobulin having a λ light chain to immature B cellsthat express an immunoglobulin having a κ light chain is about 1 toabout 13. In some other embodiments, the light chain variable domain ofthe mouse is generated through a rearrangement of a human Vκ1-39 genesegment or a human Vκ3-20 gene segment and one of two or more (e.g., 2,3, 4, or 5) human Jκ gene segments, and the ratio of mature B cells inthe bone marrow that express an immunoglobulin having a λ light chain toimmature B cells that express an immunoglobulin having a κ light chainis about 1 to about 7.

In particular embodiments, the light chain variable domain of agenetically modified mouse as described herein is generated through arearrangement of a human Vκ1-39 gene segment or a human Vκ3-20 genesegment and one of two or more (e.g., 2, 3, 4, or 5) human Jκ genesegments, and has a pro B cell population in the bone marrow within inthe range of about 2.5×10⁴ to about 1.5×10⁵ cells, inclusive, forexample about 2.5×10⁴, 3.0×10⁴, 3.5×10⁴, 4.0×10⁴, 4.5×10⁴, 5.0×10⁴,5.5×10⁴, 6.0×10⁴, 6.5×10⁴, 7.0×10⁴, 7.5×10⁴, 8.0×10⁴, 8.5×10⁴, 9.0×10⁴,9.5×10⁴, 1.0×10⁵, or 1.5×10⁵ cells; in some embodiments, a modifiedrodent (e.g., a mouse) described herein comprises a pro B cellpopulation in the bone marrow of about 2.88×10⁴ cells; in someembodiments, a modified rodent (e.g., a mouse) described hereincomprises a pro B cell population in the bone marrow of about 6.42×10⁴cells; in some embodiments, a modified rodent (e.g., a mouse) describedherein comprises a pro B cell population in the bone marrow of about9.16×10⁴ cells; in some embodiments, a modified rodent (e.g., a mouse)described herein comprises a pro B cell population in the bone marrow ofabout 1.19×10⁵ cells. Exemplary pro B cells in the bone marrow ofgenetically modified rodents (e.g., mice) as described herein arecharacterized by expression of CD19, CD43, c-kit and/or a combinationthereof (e.g., CD19⁺, CD43⁺, c-kit⁺. In some embodiments, a rodent(e.g., mouse) as described herein expresses a light chain generatedthrough a rearrangement of a human Vκ1-39 gene segment or a human Vκ3-20gene segment and one of two or more (e.g., 2, 3, 4, or 5) human Jκ genesegments, and has a pre B cell population in the bone marrow within inthe range of about 1×10⁶ to about 2×10⁶ cells, inclusive, for example,about 1.0×10⁶, 1.1×10⁶, 1.2×10⁶, 1.3×10⁶, 1.4×10⁶, 1.5×10⁶, 1.6×10⁶,1.7×10⁶, 1.8×10⁶, 1.9×10⁶, or 2.0×10⁶ cells; in some embodiments, amodified rodent (e.g., a mouse) described herein comprises a pre B cellpopulation in the bone marrow of about 1.25×10⁶ cells; in someembodiments, a modified rodent (e.g., a mouse) described hereincomprises a pre B cell population in the bone marrow of about 1.46×10⁶cells; in some embodiments, a modified rodent (e.g., a mouse) describedherein comprises a pre B cell population in the bone marrow of about1.64×10⁶ cells; in some embodiments, a modified rodent (e.g., a mouse)described herein comprises a pre B cell population in the bone marrow ofabout 2.03×10⁶ cells. Exemplary pre B cells in the bone marrow ofgenetically modified rodents (e.g., mice) as described herein arecharacterized by expression of CD19, CD43, c-kit and/or a combinationthereof (e.g., CD19⁺, CD43⁻, c-kit⁻).

In various embodiments, a genetically modified mouse as described hereinexpresses a light chain generated through a rearrangement of a humanVκ1-39 gene segment or a human Vκ3-20 gene segment and one of two ormore (e.g., 2, 3, 4, or 5) human Jκ gene segments, and has an immature Bcell population in the bone marrow within the range of about 5×10⁵ toabout 7×10⁵ cells, inclusive, for example, about 5.0×10⁵, 5.1×10⁵,5.2×10⁵, 5.3×10⁵, 5.4×10⁵, 5.5×10⁵, 5.6×10⁵, 5.7×10⁵, 5.8×10⁵, 5.9×10⁵,6.0×10⁵, 6.1×10⁵, 6.2×10⁵, 6.3×10⁵, 6.4×10⁵, 6.5×10⁵, 6.6×10⁵, 6.7×10⁵,6.8×10⁵, 6.9×10⁵, or 7.0×10⁵ cells; in some embodiments, a modifiedrodent (e.g., a mouse) described herein comprises an immature B cellpopulation in the bone marrow of about 5.33×10⁵ cells; in someembodiments, a modified rodent (e.g., a mouse) described hereincomprises an immature B cell population in the bone marrow of about5.80×10⁵ cells; in some embodiments, a modified rodent (e.g., a mouse)described herein comprises an immature B cell population in the bonemarrow of about 5.92×10⁵ cells; in some embodiments, the rodent (e.g.,mouse) comprises an immature B cell population in the bone marrow ofabout 6.67×10⁵ cells. Exemplary immature B cells in the bone marrow ofgenetically modified rodents (e.g., mice) as described herein arecharacterized by expression of IgM, B220 and/or a combination thereof(e.g., IgM⁺, B220^(int)).

In various embodiments, a genetically modified mouse as described hereinexpresses a light chain generated through a rearrangement of a humanVκ1-39 gene segment or a human Vκ3-20 gene segment and one of two ormore (e.g., 2, 3, 4, or 5) human Jκ gene segments, and has a mature Bcell population in the bone marrow within the range of about 3×10⁴ toabout 1.5×10⁵ cells, inclusive, for example about 3.0×10⁴, 3.5×10⁴,4.0×10⁴, 4.5×10⁴, 5.0×10⁴, 5.5×10⁴, 6.0×10⁴, 6.5×10⁴, 7.0×10⁴, 7.5×10⁴,8.0×10⁴, 8.5×10⁴, 9.0×10⁴, 9.5×10⁴, 1.0×10⁵, or 1.5×10⁵ cells; in someembodiments, a modified rodent (e.g., a mouse) described hereincomprises a mature B cell population in the bone marrow of about3.11×10⁴ cells; in some embodiments, a modified rodent (e.g., a mouse)described herein comprise a mature B cell population in the bone marrowof about 1.09×10⁵ cells; in some embodiments, a modified rodent (e.g., amouse) described herein comprises a mature B cell population in the bonemarrow of about 1.16×10⁵ cells; in some embodiments, a modified rodent(e.g., a mouse) described herein comprises a mature B cell population inthe bone marrow of about 1.44×10⁵ cells. Exemplary mature B cells in thebone marrow of genetically modified rodents (e.g., mice) as describedherein are characterized by expression of IgM, B220 and/or a combinationthereof (e.g., IgM⁺, B220^(hi)).

In various embodiments, a genetically modified rodent (e.g., mouse) asdescribed herein expresses a light chain generated through arearrangement of a human Vκ1-39 gene segment or a human Vκ3-20 genesegment and one of two or more (e.g., 2, 3, 4, or 5) human Jκ genesegments, and has a total B cell population in the bone marrow withinthe range of about 1×10⁶ to about 3×10⁶ cells, inclusive, for exampleabout 1.0×10⁶, 1.1×10⁶, 1.2×10⁶, 1.3×10⁶, 1.4×10⁶, 1.5×10⁶, 1.6×10⁶,1.7×10⁶, 1.8×10⁶, 1.9×10⁶, 2.0×10⁶, 2.1×10⁶, 2.2×10⁶, 2.3×10⁶, 2.4×10⁶,2.5×10⁶, 2.6×10⁶, 2.7×10⁶, 2.8×10⁶, 2.9×10⁶ or 2.0×10⁶ cells; in someembodiments, a modified rodent (e.g., a mouse) described hereincomprises a total B cell population in the bone marrow of about 1.59×10⁶cells; in some embodiments, a modified rodent (e.g., a mouse) describedherein comprises a total B cell population in the bone marrow of about1.75×10⁶ cells; in some embodiments, a modified rodent (e.g., a mouse)described herein comprises a total B cell population in the bone marrowof about 2.13×10⁶ cells; in some embodiments, a modified rodent (e.g., amouse) described herein comprises a total B cell population in the bonemarrow of about 2.55×10⁶ cells. An exemplary total B cells in the bonemarrow of genetically modified rodents (e.g., mice) as described hereinare characterized by expression CD19, CD20 and/or a combination thereof(e.g., CD19⁺).

In various embodiments, a genetically modified rodent (e.g., a mouse) asdescribed herein comprises an immunoglobulin κ light chain locus thatcomprises two unrearranged human immunoglobulin Vκ gene segments and twoor more (e.g., 2, 3, 4, or 5) unrearranged human Jκ gene segments,wherein the rodent (e.g., mouse) comprises a peripheral splenic B cellpopulation comprising transitional (e.g., T1, T2 and T3) B cellpopulations that are about the same as a rodent (e.g., a mouse) thatcomprises a wild type complement of immunoglobulin κ light chain V and Jgene segments. Exemplary transitional B cell populations (e.g., T1, T2and T3) in the spleen of a genetically modified rodent (e.g., a mouse)as described herein are characterized by expression of IgM, CD23, CD93,B220 and/or a combination thereof.

In various embodiments, a genetically modified rodent (e.g., a mouse) asdescribed herein comprises a T1 B cell population in the spleen (e.g.,CD93⁺, B220⁺, IgM^(hi), CD23⁻) within the range of about 2×10⁶ to about7×10⁶ cells, inclusive, for example about 2.0×10⁶, 2.5×10⁶, 3.0×10⁶,3.5×10⁶, 4.0×10⁶, 4.5×10⁶, 5.0×10⁶, 5.5×10⁶, 6.0×10⁶, 6.5×10⁶, or7.0×10⁶ cells; in some embodiments, a modified rodent (e.g., a mouse) asdescribed herein comprises a T1 B cell population in the spleen of about2.16×10⁶ cells; in some embodiments, a rodent (e.g., a mouse) asdescribed herein comprises a T1 B cell population in the spleen of about3.63×10⁶ cells; in some embodiments, a modified rodent (e.g., a mouse)described herein comprises a T1 B cell population in the spleen of about3.91×10⁶; in some embodiments, a modified rodent (e.g., a mouse)described herein comprises a T1 B cell population in the spleen of about6.83×10⁶ cells.

In various embodiments, a genetically modified rodent (e.g., a mouse) asdescribed herein comprises a T2 B cell population in the spleen (e.g.,CD93⁺, B220⁺, IgM^(hi), CD23⁺) within the range of about 1×10⁶ to about7×10⁶ cells, inclusive, for example about 1.0×10⁶, 1.5×10⁶, 2.0×10⁶,2.5×10⁶, 3.0×10⁶, 3.5×10⁶, 4.0×10⁶, 4.5×10⁶, 5.0×10⁶, 5.5×10⁶, 6.0×10⁶,6.5×10⁶, or 7.0×10⁶ cells; in some embodiments, a modified rodent (e.g.,a mouse) described herein comprises a T2 B cell population in the spleenof about 1.30×10⁶ cells; in some embodiments, a modified rodent (e.g., amouse) described herein comprises a T2 B cell population in the spleenof about 2.46×10⁶ cells; in some embodiments, a modified rodent (e.g., amouse) described herein comprises a T2 B cell population in the spleenof about 3.24×10⁶; in some embodiments, a modified rodent (e.g., amouse) described herein comprises a T2 B cell population in the spleenof about 6.52×10⁶ cells.

In various embodiments, a genetically modified rodent (e.g., a mouse) asdescribed herein a T3 B cell population in the spleen (e.g., CD93⁺,B220⁺, IgM^(lo), CD23⁺) within the range of about 1×10⁶ to about 4×10⁶cells, inclusive, for example about 1.0×10⁶, 1.5×10⁶, 2.0×10⁶, 2.5×10⁶,3.0×10⁶, 3.5×10⁶, or 4.0×10⁶ cells; in some embodiments, a modifiedrodent (e.g., a mouse) described herein comprises a T3 B cell populationin the spleen of about 1.08×10⁶ cells; in some embodiments, a modifiedrodent (e.g., a mouse) described herein comprises a T3 B cell populationin the spleen of about 1.35×10⁶ cells; in some embodiments, a modifiedrodent (e.g., a mouse) described herein comprises a T3 B cell populationin the spleen of about 3.37×10⁶; in some embodiments, a modified rodent(e.g., a mouse) described herein comprises a T1 B cell population in thespleen of about 3.63×10⁶ cells.

Marginal zone B cells are noncirculating mature B cells that segregateanatomically into the marginal zone (MZ) of the spleen. In rodents, MZ Bcells are sessile and reside in the outer white pulp of the spleenbetween the marginal sinus and the red pulp. This region containsmultiple subtypes of macrophages, dendritic cells, and the MZ B cells;it is not fully formed until 2 to 3 weeks after birth in rodents and 1to 2 years in humans. The MZ B cells within this region typicallyexpress high levels of sIgM, CD21, CD1, CD9 with low to negligiblelevels of sIgD, CD23, CD5, and CD11b that help to distinguish themphenotypically from follicular (FO) B cells and B1 B cells. Similar toB1 B cells, MZ B cells can be rapidly recruited into the early adaptiveimmune responses in a T cell independent manner. The MZ B cells areespecially well positioned as a first line of defense against systemicblood-borne antigens that enter the circulation and become trapped inthe spleen. It is believed they are especially reactive to bacterialcell wall components and are an important source of lipid-specificantibodies. MZ B cells also display a lower activation threshold thantheir FO B cell counterparts with heightened propensity for plasma celldifferentiation that contributes further to the accelerated primaryantibody response.

In various embodiments, a genetically modified rodent (e.g., a mouse) asdescribed herein comprising a rearranged human immunoglobulin heavychain variable region nucleotide sequence (e.g., V_(H)3-23/D/J_(H)4) hasincreased levels of marginal zone B cells relative to wild type rodents(e.g., wild type mice). In some embodiments, marginal zone B cells in agenetically modified rodent (e.g., mouse) comprising a rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence areincreased by 10%, 20%, 30%, 40%, 50% or more relative to wild typerodents (e.g., wild type mice).

In various embodiments, a genetically modified rodent (e.g., mouse) asdescribed herein comprises an immunoglobulin κ light chain locus thatcomprises two unrearranged human immunoglobulin Vκ gene segments and 1,2, 3, 4, or 5 unrearranged human immunoglobulin Jκ gene segments, andwherein the rodent (e.g., mouse) comprises a peripheral splenic B cellpopulation comprising marginal zone and marginal zone precursor B cellpopulations that are about the same as a rodent (e.g., mouse) thatcomprises a wild type complement of immunoglobulin Vκ and Jκ genesegments. Exemplary marginal zone B cell populations in the spleen of agenetically modified rodent (e.g., mouse) as described herein arecharacterized by expression of IgM, CD21/35, CD23, CD93, B220 and/or acombination thereof.

In various embodiments, a genetically modified rodent (e.g., mouse) asdescribed herein comprises marginal zone B cell population in the spleen(e.g., CD93⁻, B220⁺, IgM^(hi), CD21/35^(hi), CD23⁻) within the range ofabout 1×10⁶ to about 3×10⁶ cells, inclusive, for example, about 1.0×10⁶,1.5×10⁶, 2.0×10⁶, 2.5×10⁶, or 3.0×10⁶ cells; in some embodiments, amodified rodent (e.g., a mouse) described herein comprises a marginalzone B cell population in the spleen of about 1.47×10⁶ cells; in someembodiments, a modified rodent (e.g., a mouse) described hereincomprises a marginal zone B cell population in the spleen of about1.49×10⁶ cells; in some embodiments, a modified rodent (e.g., a mouse)described herein comprises a marginal zone B cell population in thespleen of about 2.26×10⁶ cells; in some embodiments, a modified rodent(e.g., a mouse) described herein comprises a marginal zone B cellpopulation in the spleen of about 2.33×10⁶ cells.

In various embodiments, a genetically modified rodent (e.g., mouse) isprovided, wherein the rodent (e.g., mouse) comprises an immunoglobulin κlight chain locus that comprises two unrearranged human immunoglobulinVκ gene segments and 1, 2, 3, 4, or 5 unrearranged human immunoglobulinJκ gene segments, and wherein the rodent (e.g., mouse) comprises aperipheral splenic B cell population comprising follicular (e.g., FO-Iand FO-II) B cell population(s) that are about the same as a rodent(e.g., mouse) that comprises a wild type complement of immunoglobulin Vκand Jκ gene segments. Exemplary follicular B cell populations (e.g.,FO-I and FO-II) in the spleen of a genetically modified rodent (e.g.,mouse) as described herein are characterized by expression of IgM, IgD,CD21/35, CD93, B220 and/or a combination thereof.

In various embodiments, a genetically modified rodent (e.g., mouse) asdescribed herein comprises a follicular type 1 B cell population in thespleen (e.g., CD93⁻, B220⁺, CD21/35^(int), IgM^(lo), IgD^(hi)) withinthe range of about 3×10⁶ to about 1.5×10⁷ cells, inclusive, for exampleabout 3.0×10⁶, 3.5×10⁶, 4.0×10⁶, 4.5×10⁶, 5.0×10⁶, 5.5×10⁶, 6.0×10⁶,6.5×10⁶, 7.0×10⁶, 7.5×10⁶, 8.0×10⁶, 8.5×10⁶, 9.0×10⁶, 9.5×10⁶, 1.0×10⁷,or 1.5×10⁷ cells; in some embodiments, a modified rodent (e.g., mouse)as described herein comprises a follicular type 1 B cell population inthe spleen of about 3.57×10⁶ cells; in some embodiments, a modifiedrodent (e.g., a mouse) described herein comprises a follicular type 1 Bcell population in the spleen of about 6.31×10⁶ cells; in someembodiments, a modified rodent (e.g., a mouse) described hereincomprises a follicular type 1 B cell population in the spleen of about9.42×10⁶ cells; in some embodiments, a modified rodent (e.g., a mouse)described herein comprise a follicular type 1 B cell population in thespleen of about 1.14×10⁷ cells.

In various embodiments, a genetically modified rodent (e.g., mouse) asdescribed herein comprises a follicular type 2 B cell population in thespleen (e.g., CD93⁻, B220⁺, CD21/35^(int), IgM^(int), IgD^(hi)) withinthe range of about 1×10⁶ to about 2×10⁶ cells, inclusive, for example,1.0×10⁶, 1.25×10⁶, 1.5×10⁶, 1.75×10⁶, or 2.0×10⁶ cells; in someembodiments, a modified rodent (e.g., a mouse) described hereincomprises a follicular type 2 B cell population in the spleen of about1.14×10⁶ cells; in some embodiments, a modified rodent (e.g., a mouse)described herein comprises a follicular type 2 B cell population in thespleen of about 1.45×10⁶ cells; in some embodiments, a modified rodent(e.g., a mouse) described herein comprises a follicular type 2 B cellpopulation in the spleen of about 1.80×10⁶; in some embodiments, amodified rodent (e.g., a mouse) described herein comprises a folliculartype 2 B cell population in the spleen of about 2.06×10⁶ cells.

The capabilities of the genetically modified non-human animals describedherein to apply selective pressure to genes or polynucleotides encodinglight chain variable regions or domains (e.g., light chain CDR3s) can beapplied to a variety of variable light chain gene sequences. In otherwords, the rearranged heavy chain variable domain gene sequencesdisclosed herein can be paired with one or more genetic modifications ofa light chain locus and/or the insertion of nucleotide sequencesencoding light chain variable domains into a heavy chain locus. This canbe accomplished by, for example, mating (i.e., cross-breeding orintercrossing of animals with single modification) the non-human animalsdescribed herein (restricted to a common or universal heavy chainvariable domain) with non-human animals comprising genetic modificationswithin one or more light chain-encoding loci. Genetically modifiednon-human animals comprising immunoglobulin loci with both a rearrangedheavy chain variable domain and one or more light chain modificationscan also be generated by targeted gene replacement of multiple loci,either simultaneously or sequentially (e.g., by sequential recombinationin embryonic stem cells). Neither the type nor method of modification atthe light chain loci limits embodiments described herein unlessspecifically noted. Rather, the selective pressure facilitated byembodiments described herein can be applied to virtually anypolynucleotide sequence capable of being expressed and functioning as alight chain antigen-binding sequence, thereby driving the evolution offitter antibody variable regions.

For example, as described herein, genetically modified non-human animalscomprising an immunoglobulin locus with a rearranged heavy chainvariable domain gene sequence may further comprise (e.g., viacross-breeding or multiple gene targeting strategies) one or moremodifications as described in WO 2011/072204, WO 2011/163311, WO2011/163314, WO 2012/018764, WO 2012/141798, U.S. 2013/0185821, WO2013/022782, WO 2013/096142, WO2013/116609; these publications areincorporated herein by reference in their entirety. In particularembodiments, a genetically modified mouse comprising a rearranged heavychain variable region nucleic acid sequence in a light chain locus (i.e,a rearranged heavy chain variable domain gene sequence operably linkedto a human or non-human κ light chain constant region gene sequence) iscrossed to a genetically modified mouse comprising an immunoglobulinheavy chain locus comprising human light chain variable region genesegments (e.g., 40 human Vκ genes and all human Jκ genes inserted into amouse heavy chain locus; see, e.g., U.S. pre-grant publication2012/0096572, incorporated herein by reference). In specificembodiments, a genetically modified mouse comprising a rearranged heavychain variable region nucleic acid sequence in a light chain locus (i.e,a rearranged heavy chain variable domain gene sequence operably linkedto a human or non-human κ light chain constant region gene sequence) iscrossed to a genetically modified mouse comprising an immunoglobulinheavy chain locus comprising one or more (e.g., two) but less than thewild type number of human light chain variable region gene segments. Theresulting mice are able to produce kappa+ B cells with variable heavychains derived from genomic light chain variable sequence, thusfacilitating the identification of kappa VJ sequences that bind tospecific targets, which can then be reformatted back to a light chainand paired with a variety of heavy chains to produce bi or tri specificantibodies.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use non-human animals described herein, and are not intended tolimit the scope of what the inventors regard as their invention nor arethey intended to represent that the experiments below are all or theonly experiments performed. Efforts have been made to ensure accuracywith respect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1. Cloning and Expression Analysis of Candidate Universal HeavyChain Sequences

Previous studies have shown that hV_(H)3-23 is a thermostable humanvariable heavy chain gene segment and is also one of the most commonlyused variable segments in the human repertoire. Thus, codon-optimizedhuman V_(H)3-23, D4-4 (reading frame 2 or 3), and J_(H)4 (or J_(H)6)gene segments were selected for designing a rearranged heavy chainvariable sequence (hereinafter “Universal Heavy Chain” or “UHC”).

Briefly, the following four candidate rearranged VDJ sequences weresynthesized de novo (by IDT) and cloned into CMV expression vectors(e.g., pRG1301 or pRG1368): (1) hV_(H)3-23(D4-4_RF2)J_(H)4 (SEQ ID NO:148); (2) hV_(H)3-23(D4-4_RF2)J_(H)6 (SEQ ID NO: 146); (3)hV_(H)3-23(D4-4_RF3)J_(H)4 (SEQ ID NO: 147); (4)hV_(H)3-23(D4-4_RF3)J_(H)6 (SEQ ID NO: 145). All these constructs weredesigned in a way that the synthesized UHC genes can be ligated intopRG1301 (hIgG1) or pRG1368 (mIgG1) vectors following digestion with XhoI/Sap I. For expression analysis, the four UHC genes (1-4) in pIDTSMARTwere subcloned into Xho I/Sap I sites of pRG1301 (hIgG1) or pRG1368(mIgG1), and each expression construct was transfected separately intoCHO cells. Upon transfection, all four candidate VDJ sequences wereexpressed at a sufficient level, and the expressed peptides were capableof pairing with different K and A light chains.

In order to avoid potential autoreactive antibodies that might lead to Bcell depletion in the genetically modified mice, the ASAP antibodydatabase of Regeneron Pharmaceuticals, which was generated from theantibodies produced by VELOCIMMUNE® humanized mice, was searched forantibodies containing an amino acid sequence that is similar tohV_(H)3-23(D4-4)J_(H)4 (FIG. 5). More specifically, the criteria thatwere used to identify non-autoreactive antibodies included the aminoacid sequence of DYSNY (SEQ ID NO: 144) or sequences similar to DYSNY(SEQ ID NO: 144). Expression studies in CHO cells, however, revealedthat UHC sequences containing DYSNY (SEQ ID NO: 144) did not expresswell in mammalian cells. Therefore, another sequence that lacks the Dbut has the sequence YSNY (i.e., antibody H1H2002B; AKGYYFDY (SEQ ID NO:143); wherein AK is from 3-23; GY is from a D or an N addition or N andP additions; and YFDY is J_(H)4) was selected instead and tested for itsexpression in CHO cells. The modified UHC sequence was expressed at asufficient level in CHO cells. These results suggested that some aminoacid residues (i.e., a spacer) are required between the sequence encodedby a heavy chain V gene segment and the sequence encoded by a heavychain J gene segment for proper expression of the rearranged VDJsequence in mammalian cells.

In addition, the expression levels of the peptide AKGYYFDY derived fromthe rearranged VDJ sequence (V_(H)3-23/GY/J_(H)4; HIH2002B) in CHO cellswere compared with the peptide derived from of V_(H)3-23/D4-4 (readingframe 2)/J_(H)4 (SEQ ID NO: 148), with respect to expression with fivehuman κ chains, three human λ chains, and other rearranged VDJ sequences(i.e., V_(H)3-20 and V_(H)1-39). The selected rearranged VDJ sequence(V_(H)3-23/GY/J_(H)4) showed expression levels equivalent to those ofthe controls.

Based on these data, V_(H)3-23/GY/J_(H)4 (SEQ ID NO: 137; HIH2002B) wasselected as a rearranged heavy chain variable domain sequence forcreating a genetically modified mouse. Detailed targeting strategies forgenerating a mouse containing a genetically modified immunoglobulinlocus that encodes a rearranged heavy chain variable domain (i.e., amouse that comprises an immunoglobulin locus comprising a rearrangedhuman immunoglobulin heavy chain variable region) are illustrated inFIGS. 1-9 and as described below.

Example 2. Construction of Immunoglobulin Heavy Chain Loci Containing aRearranged VDJ Sequence

Construction of immunoglobulin heavy chain loci containing a rearrangedhuman VDJ sequence was carried out by series of homologous recombinationreactions in bacterial cells (BHR) using Bacterial Artificial Chromosome(BAC) DNA. Several targeting constructs for creation of a geneticallyengineered mouse that expresses the rearranged heavy chain variabledomain were generated using VELOCIGENE® genetic engineering technology(see, e.g., U.S. Pat. No. 6,586,251 and Valenzuela, D. M. et al. (2003),High-throughput engineering of the mouse genome coupled withhigh-resolution expression analysis, Nature Biotechnology 21(6):652-659,incorporated herein by reference in their entireties).

Briefly, targeting vectors were designed to introduce a rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence (i.e.,hV_(H)3-23(D)J_(H)4; SEQ ID NO: 136) into a genetically modified mousein which all or substantially all endogenous functional immunoglobulinheavy chain V, D, J gene segments have been deleted. In addition, thetargeting vectors included a genomic region comprising Adam6a and Adam6bgenes in order to prevent fertility problems associated with thedeletion of the genomic region comprising Adam6a/6b genes in mice (see,for example, US 2012-0322108A1, incorporated by reference herein in itsentirety).

Initially, a BHR donor for modifying a mouse BAC clone comprising aleader sequence (which guides the heavy chain through the endoplasmicreticulum), a rearranged heavy chain variable region nucleotide sequence(V_(H)3-23(D)J_(H)4; SEQ ID NO: 136) and an intron of hJ_(H)4 (SEQ IDNO: 140) that are operably linked to a 2239-bp V_(H)3-23 promoter (SEQID NO: 139), was constructed. Additionally, the genomic locus wasflanked 5′ and 3′ by mouse IgH homology boxes for homologousrecombination with the MAID1115 BAC clone (FIG. 1).

In addition, the following five modifications have been carried out tocreate a targeting construct containing a rearranged humanimmunoglobulin heavy chain variable region nucleotide sequence.

First, a spectinomycin selection cassette was introduced into theupstream of the V_(H)3-23 promoter (between the I-CeuI and SpeI sites)to generate pJSh0038 (UHC mini-locus; SEQ ID NO: 142) (FIG. 1, 1.I-CeuI/SpeI Ligation (Amp+Spec)). The UHC mini-locus contains: (1) aspectinomycin (Spec) cassette with I-CeuI/AscI sites for ligation; (2)2239-bp hV_(H)3-23 promoter (SEQ ID NO: 139); (3) a rearrangedhV_(H)3-23(D)J_(H)4 nucleotide sequence (SEQ ID NO: 136); (4) an hJ_(H)4intron (SEQ ID NO: 140); and (5) mouse homology boxes for BHR (MAID1115).

Second, a hygromycin selection cassette (EM7-HYG) was targeted into the5′ end of the genomic region of the MAID 1115 BAC clone, which containsa loxP-flanked neomycin cassette (Pgk-Neo) in the upstream of the IgMgenomic region. Insertion of the hygromycin cassette deleted the loxPsite located at the 5′ end of the MAID 1115 clone. The bacterial cellscontaining the genetically modified BAC clone (VI432) were selected viahygromycin/kanamycin selection (FIG. 2, 2. BHR (Hyg+Kan)).

Third, the UHC mini-locus, which was constructed in Step 1, was targetedinto the upstream of the IgM locus of the VI432 BAC clone. Theintroduction of the UHC mini-locus replaced the floxed neomycinselection cassette with a new spectinomycin cassette (VI443). Bacterialcells containing the genetically modified BAC clones (VI443) wereselected via spectinomycin and hygromycin selection (FIG. 2; 3. BHR(Spec+Hyg)).

Fourth, the VI421 BAC clone, which comprises, from 5′ to 3′, (1) anAdam6a gene (present in a 3′ to 5′ direction); (2) a neomycin cassette(present in a 3′ to 5′ direction) flanked by FRT sites; (3) an Adam6bgene (present in a 3′ to 5′ direction); (4) Intergenic Control Region 1(IGCR1; i.e., a key V(D)J recombination regulatory region); and (5) aspectinomycin cassette (present in a 5′ to 3′ direction), were targetedwith the pDBa0049 construct, which contains a chloramphenicol (Cm)cassette; an AscI restriction site upstream of the chloramphenicol gene;and 5′ and 3′ homology arms. The targeting of the pDBa0049 constructremoved IGCR1 and the spectinomycin cassette from the VI421 clone; andintroduced a new AscI restriction site and a chloramphenicol cassette tothe downstream of the Adam6b gene. Bacterial cells containing thesuccessfully targeted clone (VI444) were selected via chloramphenicoland kanamycin selection (FIG. 3; 4. BHR (Cm+Kan)).

Fifth, the genomic region of the VI444 BAC clone containing the Adam6aand/or 6b genes were introduced into the upstream of the universal heavychain genomic locus in the VI443 BAC clone between the I-CeuI and theAscI sites via restriction digestion and ligation (FIG. 3). Thismodification introduces Adam6a and/or 6b genes into the clone andreplaces the spectinomycin cassette with a neomycin cassette, yielding afinal targeting construct (MAID6031; VI445). The bacterial cells (BHR)containing the final targeting construct (MAID 6031; VI445) wereselected based on hygromycin and kanamycin selection (FIG. 3, 5.I-CeuI/AscI ligation (Hyg+Kan)).

The final targeting construct (MAID6031) for the creation of a genomiclocus containing a rearranged human heavy chain variable domain sequencecontains, from 5′ to 3′, (1) a 5′ homology arm containing about 20000 bpof a mouse genomic sequence upstream of the endogenous Ig heavy chainlocus; (2) an Adam6a gene; (3) a 5′ FRT site; (4) a neomycin cassette;(5) a 3′ FRT site, (6) an Adam6b gene; (7) 2239 bp of hVH3-23 promoter(SEQ ID NO: 139); (8) a rearranged human immunoglobulin heavy chainnucleotide sequence (hVH3-23(D)J_(H)4; SEQ ID NO: 136); (9) an hJ_(H)4intron (SEQ ID NO: 140); and (10) a 3′ homology arm containing about7400 bp of a mouse genomic sequence downstream of the mouse J_(H) genesegments.

The final targeting construct, MAID6031 BAC DNA, was linearized andelectroporated into ES cells isolated from the 1661 heterozygous mouse(FIG. 4), which contain a wild-type Ig heavy chain VDJ genomic loci anda mutated VDJ genomic loci in which all V_(H), D, J_(H) genes have beendeleted. Successfully targeted mouse ES cells were screened using theprimers and probes set forth in FIGS. 6-8. The successfully targetedmouse ES cells were introduced into host mouse embryos usingVELOCIMOUSE® technology to produce a genetically modified heterozygousFO mouse. In order to generate mice (MAID 6032 het) without theselection cassette (i.e., FRT-Ub-Neo-FRT), the successfully targeted EScells were electroporated with a plasmid that expresses Flp recombinaseprior to introducing into host embryos. Alternatively, MAID6031heterozygous male mice harboring the selection cassette were bred tofemale mice that express Flp recombinase in order to remove thecassette. Heterozygous mice bearing the modification were bred to eachother to generate homozygotes (MAID 6032 HO) that are capable of makingimmunoglobulin heavy chains only from the genetically modified locus.

Example 3. Characterization of Genetically Modified Mice Expressing aRearranged Heavy Chain Variable Domain

All mice were housed and bred in specific pathogen-free conditions atRegeneron Pharmaceuticals. Three wild type (WT) littermate control mice(16 weeks old, male, n=2; Background: 75% C57/BL6 and 25% 129) and twoto four MAID 6032 HET FO mice (FIG. 9; 9 weeks old, male, n=2;Background: 50% C57/BL6 and 50% 129) were sacrificed, and blood, spleensand bone marrow were harvested from the animals. Additionally, four wildtype (WT) littermate control mice (10 weeks old; 2 male and 2 female)and four MAID 6032 homozygous (“HO”) F2 mice (10 weeks old; 3 male; 1female) were sacrificed, and blood, spleens and bone marrow wereharvested from the animals. Blood was collected into BD microtainertubes with EDTA (Cat #365973). Bone marrow was collected from femurs byflushing with complete RPMI medium (RPMI medium supplemented with fetalcalf serum, sodium pyruvate, Hepes, 2-mercaptoethanol, non-essentialamino acids, and gentamycin). Red blood cells from peripheral blood,spleen and bone marrow preparations were lysed with ACK lysis buffer andwashed with complete RPMI medium.

Flow Cytometry

In order to examine the ability of the genetically modified heterozygousFO mice (MAID 6032 HET) described herein to produce antibodies derivedfrom the genetically modified allele (i.e., from the allele thatcontains a single copy of the rearranged V_(H)3-23/D/J_(H)4),fluorescence-activated cell sorting (FACS) analysis was performed usingblood, spleen, bone marrow cells isolated from a wild-type or a 6032heterozygous mouse.

Briefly, 1×10⁶ cells were incubated with mouse anti-CD16/CD32 antibodies(2.4G2, BD) on ice for 10 minutes, followed by labeling with thefollowing antibody cocktail for 30 min on ice: anti-mouse FITC-IgM^(a)(DS-1, BD Biosciences), Pacific blue-CD3 (17A2, BioLegend), APC-H7-CD19(1 D3, BD Biosciences) and PE-IgM^(b) (AF6-78, BioLegend). Stained cellswere washed and fixed in 2% formaldehyde. Data acquisition was performedon the BD LSRFortessa flow cytometer and analyzed with FlowJo. Bonemarrow cells, spleen cells, and blood cells isolated from a wild type orFO 6032 heterozygous mouse were gated on singlets and sorted based onCD19 expression (a B cell marker) or CD3 expression (a T cell marker).In addition, CD19+-gated B cells were sorted based on the presence ofIgM^(b) antibodies (IgM antibodies produced from a wild type allele (B6allele)) or IgM^(a) antibodies (antibodies produced from the geneticallymodified allele (129 allele) comprising a rearranged heavy chainvariable region nucleotide sequence (hV_(H)3-23(D)J_(H)4). The FACSanalysis (FIGS. 11-12) suggested that the mice heterozygous with respectto the targeted allele (i.e., containing one copy of the rearrangedheavy chain variable sequence; MAID 6032 het) were able to produce IgMantibodies mostly derived from the genetically modified 129 (IgM^(a))allele.

In order to examine the ability of the genetically modified homozygousF2 mice (MAID 6032 HO) described herein to produce antibodies derivedfrom the genetically modified allele (i.e., from the allele thatcontains a single copy of the rearranged V_(H)3-23/D/J_(H)4),fluorescence-activated cell sorting (FACS) analysis was performed asdescribed above using spleen and bone marrow cells isolated from awild-type or a 6032 homozygous mouse.

Only mature B lymphocytes can enter the lymphoid follicles of spleen andlymph nodes and thus efficiently participate in the immune response.Mature, long-lived B lymphocytes derive from short-lived precursorsgenerated in the bone marrow. Selection into the mature pool is anactive process and takes place in the spleen. Two populations of splenicB cells have been identified as precursors for mature B cells.Transitional B cells of type 1 (T1) are recent immigrants from the bonemarrow. They develop into the transitional B cells of type 2 (T2), whichare cycling and found exclusively in the primary follicles of thespleen. Mature B cells can be generated from T1 or T2 B cells. Loder, F.et al., J. Exp. Med., 190(1): 75-89, 1999.

The FACS analysis (FIGS. 13A and 13B) suggested that the mice homozygouswith the respect to the targeted allele (i.e., containing two copies ofthe rearranged heavy chain variable sequence: MAID 6032 HO) were able toproduce normal splenic mature and immature B cell populations, albeitwith a slight decrease in the lambda sequences relative to wild type(FIGS. 13C and 13D). Also in the spleen, the MAID 6032HO micedemonstrated a slight decrease in T1 population B cells and an increasein marginal zone B cells (FIG. 13E).

In the bone marrow, the MAID6032 HO mice produced near normal B cellpopulations (FIGS. 14A-14E) with a usage of lambda sequences that washalf of wild type (FIG. 14F).

Immunization Studies

Five WT (75% C57BL6/25% 129 background) and three to four MAID 6032 HETmice were immunized in the footpad with 0.025 ml of a mixture containing2.35 μg of an antigen X, 10 μg CpG oligonucleotide (ODN 1826, InvivoGen,cat #tlrl-1826), and 25 μg Aluminum Phosphate Gel Adjuvant (Brenntag cat#7784-30-7). Mice were boosted six times with the same dosage. On days15 and 24 post primary immunization, blood was collected fromanaesthetized mice using a retro-orbital bleed into BD serum separatortubes (BD, cat #365956), and serum was collected as per manufacturer'sdirections.

To measure the levels of antigen-specific IgG antibodies and tocounterscreen the mmh (myc-myc-his) tag, ELISA plates (Nunc) were coatedwith either 1 μg/ml of an antigen X incubated overnight at 4 deg C.Excess antigen was washed off before blocking with PBS+1% BSA for 1 hrat RT. Serial dilutions of serum were applied and plates were incubatedfor 1 hr at RT before washing. Plates were incubated with horseradishperoxidase (HRP)-conjugated anti-IgG (cat #1030-05, Southern Biotech)antibody for 1 hr at RT. Following washing, plates were developed withTMB substrate (cat #555214, BD). Reactions were stopped with 1N sulfuricacid, and O.D. was read at 450 nm, using a Victor X5 Perkin ElmerReader. Data was analyzed with GraphPad Prism to calculate the dilutionof serum that falls two times above background. All animal experimentswere approved by IACUC and Regeneron Pharmaceuticals.

As shown in FIG. 15, the genetically modified FO and F1 mice (MAID 6032het), which are heterozygous with respect to the targeted allele (i.e.,containing one copy of the rearranged V_(H)3-23/D/J_(H)4 nucleotidesequence), were able to produce antigen-specific IgG antibodies atlevels comparable to those produced by wild type mice at both Days 15and 24 post primary immunization.

Example 3. Generation and Analysis of Mice Comprising Two Human LightChain V Segments Example 3.1: Construction of Targeting Vector forGeneration of Mice that Comprise Two Human Light Chain V Segments

Two engineered light chain loci containing two human Vκ gene segments(e.g., a human Vκ1-39 and human Vκ3-20 gene segment; i.e., a dual lightchain (“DLC”)) were constructed (FIG. 20). One engineered light chainlocus contained two human Vκ gene segments and five human Jκ genesegments in unrearranged configuration (DLC-5J). The second engineeredlight chain locus contained two human Vκ gene segments and one human Jκgene segment in unrearranged configuration (DLC-1J). For each of the twoadditional engineered light chain loci, the human gene segments wereflanked 3′ with recombination signal sequences to allow for in vivorearrangement of the human gene segments in B cells.

Engineering and Generation of DLC-1J Mice. Engineering steps that resultin generation of a light chain locus comprising two human Vκ genesegments (Vκ1-39 and Vκ3-20) and one human Jκ gene segment (Jκ5),otherwise termed as DLC-1J, are depicted in FIG. 21. Specifically, humanVκ1-39 and Vκ3-20 sequences were amplified by PCR from BAC templates(Invitrogen), and together with an amplified sequence containingrecombination signal sequence (rss) and human Jκ5 segment, cloned via afour-way ligation into a plasmid containing a UB-hygromycin selectioncassette (FIG. 21A). 5′ and 3′ arms were attached as depicted in FIGS.21B and 21C.

The resultant targeting construct is depicted in FIG. 21C (bottomdiagram; DLC-1J), with recombination signal sequences (RSS) in clearovals. Modified BAC DNA clone of the engineered DLC-1J light chain locusoperably linked to mouse sequences (i.e., upstream and downstreamsequences of the endogenous immunoglobulin κ light chain locus) wasconfirmed by PCR using primers located at sequences within theengineered light chain locus containing the two human Vκ gene segments,followed by electroporation into ES cells comprising deletion of themouse Igκ variable locus (comprising K variable and joining genesegments) (FIG. 21D) to create a mouse that expresses either of the twohuman Vκ gene segments. Positive ES cell clones that contained theengineered DLC-1J light chain locus was confirmed by Taqman™ screeningand karyotyping using probes specific for the engineered DLC-1J lightchain locus. Sequences of primers and probes used for ES cell screeningof DLC-1J ES cells are depicted in Table 8 below and are included inSequence Listing.

TABLE 8 Primers and Probes Used for ES Cell Screening Probe Assay/typeLocation Probe Forward Reverse Name of probe detected Sequence PrimerPrimer 1633h2 GOA/ Vκ1-39 ATCAGCAGAA GGGCAAG TGCAAACTGG TAQMAN™ACCAGGGAAA TCAGAGC ATGCAGCATA GCCCCT ATTAGCA G (SEQ ID (SEQ ID (SEQ IDNO: 46) NO: 44) NO: 45 1635h2 GOA/ Vκ3-20 AAAGAGCCAC TCCAGGC AAGTAGCTGCTAQMAN™ CCTCTCCTGC ACCCTGTC TGCTAACACT AGGG TTTG CTGACT  (SEQ ID (SEQ ID(SEQ ID NO: 65) NO: 66)  NO:67) Neo GOA neo TGGGCACAAC GGTGGAG GAACACGGCAGACAATCGG AGGCTATT GGCATCAG CTG CGGC (SEQ ID (SEQ ID (SEQ ID NO: 40)NO: 38) NO: 39) Jxn 1-39/ GOA/BHQ1 1-39/ TCTTTTGCCCC GGGAGGC GTCCAGTCAC3-20 3-20 GGATCCGATC TCCTCTGA TCGGTTGCTA BamHI AG (SEQ ID ACTCTAAG Tjunction NO: 84; (SEQ ID (SEQ ID restriction  NO: 85) NO: 86)site bolded)

Confirmed ES cell clones were then used to implant female mice to giverise to a litter of pups comprising DLC-1J light chain locus andexpressing a human light chain variable domain fused with a mouse Cκdomain. Sequences of primers and probes used for genotyping of the pupsare listed in Table 8 above. The sequence through the engineered DLC-1Jlocus, including 100 nucleotides of mouse sequence upstream anddownstream of the inserted engineered sequence is presented in FIGS.22A-22D and set forth in SEQ ID NO:82.

ES cells bearing the engineered light chain locus may be transfectedwith a construct that expresses FLP in order to remove the FRTedneomycin cassette introduced by the targeting construct (see FIG. 21E).Optionally, the neomycin cassette is removed by breeding to mice thatexpress FLP recombinase (e.g., U.S. Pat. No. 6,774,279). Optionally, theneomycin cassette is retained in the mice.

Engineering and Generation of DLC-5J Mice. To generate a light chainlocus comprising two human Vκ gene segments (Vκ1-39 and Vκ3-20) and fivehuman Jκ gene segments (Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5), otherwise termedas DLC-5J, a 2000 base pair amplified sequence comprising all 5 humanJκ's was ligated into a vector comprising two human Vκ gene segments andone human Jκ, depicted in FIG. 21B (middle) (see FIG. 23A). Subsequentengineering steps involved attachment of 3′ and 5′ arms as depicted inFIG. 23B.

The resultant targeting construct is depicted in FIG. 23B (bottomdiagram; DLC-5J), with recombination signal sequences (RSS) in clearovals. Modified BAC DNA clone the engineered DLC-5J light chain locusoperably linked to mouse sequences (i.e., upstream and downstreamsequences of the endogenous immunoglobulin κ light chain locus) wasconfirmed by PCR using primers located at sequences within theengineered light chain locus containing the two human Vκ gene segments,followed by electroporation into ES cells comprising deletion of themouse Igκ variable locus (comprising K variable and joining genesegments) (FIG. 23C) to create a mouse that expresses either of the twohuman Vκ gene segments. Positive ES cell clones that contained theengineered DLC-5J light chain locus was confirmed by Taqman™ screeningand karyotyping using probes specific for the engineered DLC-5J lightchain locus. Sequences of primers and probes used for ES cell screeningof DLC-5J ES cells are depicted in Table 9 below and are included inSequence Listing.

TABLE 9  Primers and Probes Used for ES Cell Screening Probe Assay/typeLocation Probe Forward Reverse Name of probe detected Sequence PrimerPrimer 1633h2 GOA/ Vκ1-39 ATCAGCAGAA GGGCAAG TGCAAACTGG TAQMAN™ACCAGGGAAA TCAGAGC ATGCAGCATA GCCCCT ATTAGCA G (SEQ ID (SEQ ID (SEQ IDNO: 44) NO: 45 NO: 46) 1635h2 GOA/ Vκ3-20 AAAGAGCCAC TCCAGGC AAGTAGCTGCTAQMAN™ CCTCTCCTGC ACCCTGTC TGCTAACACT AGGG TTTG CTGACT (SEQ ID (SEQ ID(SEQ ID  NO: 65) NO: 66) NO: 67) Neo GOA neo TGGGCACAAC GGTGGAGGAACACGGC AGACAATCGG AGGCTATT GGCATCAG CTG CGGC (SEQ ID (SEQ ID (SEQ IDNO: 40) NO: 38) NO: 39) Jxn 1-39/ GOA/BHQ1 1-39/3-20 TCTTTTGCCCC GGGAGGCGTCCAGTCAC 3-20 BamHI GGATCCGATC TCCTCTGA TCGGTTGCTA junction AG (SEQ IDACTCTAAG T (SEQ ID NO: 84; (SEQ ID NO: 86) restriction NO: 85) sitebolded) Jxn 3- G0A/BHQ1 3-20/Jk1-5 CTTCAACTGTG ACGCAGA CAGCTGCTGA20/Jk1-5 BsiWI GCGTACGCAC TGTAGCCA AGCTCAACTC junction C (SEQ ID AACCCT(SEQ ID NO: 87, (SEQ ID NO: 89) restriction NO: 88) site bolded)

Confirmed ES cell clones were then used to implant female mice to giverise to a litter of pups comprising DLC-5J light chain locus andexpressing a human light chain variable domain fused with a mouse Cκdomain. Sequences of primers and probes used for genotyping of the pupsare listed in Table 9 above. The sequence through the engineered DLC-5Jlocus, including 100 nucleotides of mouse sequence upstream anddownstream of the inserted engineered sequence is presented in FIGS.24A-24D and set forth in SEQ ID NO:83.

ES cells bearing the engineered light chain locus may be transfectedwith a construct that expresses FLP in order to remove the FRTedneomycin cassette introduced by the targeting construct (see FIG. 23D).Optionally, the neomycin cassette is removed by breeding to mice thatexpress FLP recombinase (e.g., U.S. Pat. No. 6,774,279). Optionally, theneomycin cassette is retained in the mice.

Example 3.2: Characterization of Mice that Comprise Two Human V Segments

Flow Cytometry. B cell populations and B cell development in DLC micewere validated by flow cytometry analysis of splenocyte and bone marrowpreparations. Cell suspensions from mice homozygous for two human Vκgene segments and five human Jκ gene segments (n=4), mice homozygous fortwo human Vκ gene segments and one human Jκ gene segment (n=4), and wildtype mice (n=4) were made using standard methods and stained withfluorescently labeled antibodies.

Briefly, 1×10⁶ cells were incubated with anti-mouse CD16/CD32 (clone2.4G2, BD Pharmigen) on ice for 10 minutes, followed by labeling withthe following antibody cocktail for 30 minutes on ice: APC-H7 conjugatedanti-mouse CD19 (clone 1D3, BD Pharmigen), Pacific Blue conjugatedanti-mouse CD3 (clone 17A2, BioLegend), FITC conjugated anti-mouse Igκ(clone 187.1, BD Pharmigen) or anti-mouse CD43 (clone 1B11, BioLegend),PE conjugated anti-mouse Ig A (clone RML-42, BioLegend) or anti-mousec-kit (clone 2B8, BioLegend), PerCP-Cy5.5 conjugated anti-mouse IgD(BioLegend), PE-Cy7 conjugated anti-mouse IgM (clone II/41,eBioscience), APC conjugated anti-mouse B220 (clone RA3-6B2,eBioscience). Following staining, cells were washed and fixed in 2%formaldehyde. Data acquisition was performed on an LSRII flow cytometerand analyzed with FlowJo (Tree Star, Inc.). Gating: total B cells(CD19+CD3−), Igκ+ B cells (Igκ+Ig_(λ)−CD19+CD3−), Ig_(λ)+ B cells(Igκ−Ig_(λ)+CD19+CD3−). Results for the bone marrow compartment areshown in FIG. 25A-27B. Results for the splenic compartment are shown inFIG. 28A-FIG. 31.

As shown in this Example, DLC-5J mice demonstrate normal B cellpopulations within the splenic and bone marrow compartments (FIG.25A-31). DLC-5J mice demonstrated immature, mature and pre/pro B cellpopulations within the bone marrow compartment that are substantiallythe same as observed in wild-type littermates. In fact, the DLC-5J locuswas capable of competing with the endogenous lambda light chain locus toyield a kappa:lambda ratio that is substantially the same as thatobserved in wild-type mice (FIG. 27B). Also, DLC-5J mice demonstrate anormal peripheral B cell development as progression of B cells throughvarious stages in the splenic compartment (e.g., immature, mature, T1,T2 T3, marginal zone precursor, marginal zone, follicular-I,follicular-II, etc.) occurs in a manner substantially the same asobserved in wild type mice (FIG. 30A-31). In contrast, DLC-1J micedemonstrated a lower overall number of B cells and an increased lambdalight chain usage as compared to the engineered kappa light chain (datanot shown).

Dual Light Chain Expression. Expression of both human Vκ gene segmentswas analyzed in homozygous mice using a quantitative PCR assay. Briefly,CD19+ B cells were purified from bone marrow and whole spleens of wildtype mice, mice homozygous for a replacement of the mouse heavy chainand κ light chain variable loci with corresponding human heavy chain andκ light chain variable region loci (HK), as well as mice homozygous foran engineered κ light chain loci containing two human Vκ gene segmentsand either five human Jκ gene segments (DLC-5J) or one human Jκ genesegment (DLC-1J). Relative expression was normalized to expression ofmouse Cκ region (n=3 to 5 mice per group). Results are shown in FIG. 32and FIG. 33.

Expression of light chains containing a rearranged human Vκ3-20 or humanVκ1-39 gene segment were detected in both the bone marrow and spleen ofDLC-5J and DLC-1J mice (FIG. 32 and FIG. 33). In the bone marrowcompartment, expression of both human Vκ3-20-derived and humanVκ1-39-derived light chains in both strains of DLC mice wassignificantly higher as compared to mice comprising a replacement ofmouse Vκ and Jκ gene segment with corresponding human Vκ and Jκ genesegments (HK; FIG. 32). Human Vκ3-20-derived light chain expression wasobserved at about six-fold (DLC-5J) to fifteen-fold (DLC-1J) higher thanin HK mice. DLC-1J mice demonstrated about two-fold higher expression ofhuman Vκ3-20-derived light chains over DLC-5J mice in the bone marrowcompartment. Human Vκ1-39-derived light chain expression was observed atabout six-fold (DLC-5J) to thirteen-fold (DLC-1J) higher than in HKmice. DLC-1J mice demonstrated about two-fold higher expression of humanVκ1-39-derived light chains over DLC-5J mice in the bone marrowcompartment.

In the splenic compartment, expression of both human Vκ3-20-derived andhuman Vκ1-39-derived light chains in both strains of DLC mice wassignificantly higher as compared to HK mice (FIG. 33). HumanVκ3-20-derived light chain expression was observed at about four-fold(DLC-5J) and eight-fold (DLC-1J) higher than in HK mice. DLC-1J micedemonstrated about two-fold higher expression of human Vκ3-20-derivedlight chains over DLC-5J mice in the splenic compartment. HumanVκ1-39-derived light chain expression was observed at about four-fold(DLC-5J) to five-fold (DLC-1J) higher than in HK mice. DLC-1J micedemonstrated similar expression of human Vκ1-39-derived light chains ascompared to DLC-5J mice in the splenic compartment.

Human Vκ/Jκ Usage in DLC-5J Mice. Mice homozygous for two unrearrangedhuman Vκ gene segments and five unrearranged human Jκ gene segments(DLC-5J) were analyzed for human Vκ/Jκ gene segment usage in splenic Bcells by reverse-transcriptase polymerase chain reaction (RT-PCR).

Briefly, spleens from homozygous DLC-5J (n=3) and wild type (n=2) micewere harvested and meshed in 10 mL of RPMI 1640 (Sigma) containing 10%heat-inactivated fetal bovine serum using frosted glass slides to createsingle cell suspensions. Splenocytes were pelleted with a centrifuge(1200 rpm for five minutes) and red blood cells were lysed in 5 mL ofACK lysing buffer (GIBCO) for three minutes. Splenocytes were dilutedwith PBS (Irvine Scientific), filtered with a 0.7 μm cell strainer andcentrifuged again to pellet cells, which was followed by resuspension in1 mL of PBS.

RNA was isolated from pelleted splenocytes using AllPrep DNA/RNA minikit (Qiagen) according to manufacturer's specifications. RT-PCR wasperformed on splenocyte RNA using 5′ RACE (Rapid Amplification of cDNAends) System with primers specific for the mouse Cκ gene according tomanufacturer's specifications (Invitrogen). The primers specific for themouse Cκ gene were 3′ mIgκC RACE1 (AAGAAGCACA CGACTGAGGC AC; SEQ ID NO:90) and mIgκC3′-1 (CTCACTGGAT GGTGGGAAGA TGGA; SEQ ID NO: 91). PCRproducts were gel-purified and cloned into pCR®2.1-TOPO® vector (TOPO®TA Cloning® Kit, Invitrogen) and sequenced with M13 Forward (GTAAAACGACGGCCAG; SEQ ID NO: 92) and M13 Reverse (CAGGAAACAG CTATGAC; SEQ ID NO:93) primers located within the vector at locations flanking the cloningsite. Ten clones from each spleen sample were sequenced. Sequences werecompared to the mouse and human immunoglobulin sets from theIMGT/V-QUEST reference directory sets to determine Vκ/Jκ usage. Table 10sets forth the Vκ/Jκ combinations for selected clones observed in RT-PCRclones from each splenocyte sample. Table 11 sets forth the amino acidsequence of the human Vκ/human Jκ and human Jκ/mouse Cκ junctions ofselected RT-PCR clones from DLC-5J homozygous mice. Lower case lettersindicate mutations in the amino acid sequence of the variable region ornon-template additions resulting from N and/or P additions duringrecombination.

As shown in this example, mice homozygous for two unrearranged human Vκgene segments and five unrearranged human Jκ gene segments (DLC-5J)operably linked to the mouse Cκ gene are able to productively recombineboth human Vκ gene segments to multiple human Jκ gene segments toproduce a limited immunoglobulin light chain repertoire. Among therearrangements in DLC-5J homozygous mice shown in Table 10, unique humanVκ/Jκ rearrangements were observed for Vκ1-39/Jκ2 (1), Vκ1-39/Jκ3 (1),Vκ3-20/Jκ1 (7), Vκ3-20/Jκ2 (4) and Vκ3-20/Jκ3 (1). Further, such uniquerearrangements demonstrated junctional diversity through the presence ofunique amino acids within the CDR3 region of the light chain (Table 11)resulting from either mutation and/or the recombination of the human Vκand Jκ gene segments during development. All the rearrangements showedfunctional read through into mouse Cκ (Table 11).

Taken together, these data demonstrate that mice engineered to present achoice of no more than two human Vκ gene segments, both of which arecapable of rearranging (e.g., with one or more and, in some embodiments,up to five human Jκ gene segments) and encoding a human V_(L) domain ofan immunoglobulin light chain have B cell numbers and development thatis nearly wild-type in all aspects. Such mice produce a collection ofantibodies having immunoglobulin light chains that have one of twopossible human V_(L) gene segments present in the collection. The mouseproduces this collection of antibodies in response to antigen challengeand, and the collection of antibodies is associated with a diversity ofreverse chimeric (human variable/mouse constant) heavy chains.

TABLE 10 Vκ/Jκ Combinations Observed in Splenocyte Samples Mouse ID No.Genotype Clone Vκ/Jκ Combination 1089451 DLC-5J 1-2 1-39/3 1-4 3-20/21-7 3-20/1 1-8 3-20/2 1089452 DLC-5J 2-2 3-20/1 2-3 3-20/1 2-6 3-20/22-8 3-20/2 2-9 3-20/1 2-10 1-39/2 1092594 DLC-5J 3-1 3-20/1 3-2 3-20/13-4 3-20/1 3-6 3-20/3 3-9 3-20/2 1092587 WT 1-1 19-93/1  1-2 6-25/1 1-34-91/5 1-5 3-10/4 1-6 4-86/4 1-8 19-93/1  1-10 19-93/2  1092591 WT 2-119-93/1  2-3 6-20/5 2-4 6-25/5 2-5 1-117/1  2-6 8-30/1 2-7 8-19/2 2-88-30/1 2-10 1-117/1 

TABLE 11 Amino Acid Sequences of the Human Vκ/HumanJκ and Human Jκ/Mouse Cκ Junctions from DLC-5J Homozygous MiceSequence of hVκ/hJκ/mCκ Junction SEQ (CDR3 underlined, ID Clone Vκ/JκmIgκC italics) NO: 2-10 1-39/2 QPEDFATYYCQQSYSTPYTF 94GQGTKLEIKRADAAPTVSI 1-2 1-39/3 QPEDFATYYCQQSYSTPFTF 95IGPGTKVDIKRADAAPTVS 1-7 3-20/1 EPEDFAVYYCQQYGSSPrTF 96GQGTKVEIKRADAAPTVSI 2-2 3-20/1 EPEDFAVYYCQQYGSSrTF 97GQGTKVEIKRADAAPTVSI 2-3 3-20/1 EPEDFAVYYCQQYGSSPWTF I 98GQGTKVEIKRADAAPTVS 2-9 3-20/1 dPEDFAVYYCQQYGSSPrTF 99GQGTKVEIKRADAAPTVSI 3-1 3-20/1 EPEDFAVYYCQQYGSSPrTF 100GQGTKVEIKRADAAPTVSI 3-2 3-20/1 EPEDFAVYYCQQYGSSPWTF 101GQGTKVEIKRADAAPTVSI 3-4 3-20/1 EPEDFAVYYCQQYGSSPPTF 102GQGTKVEIKRADAAPTVSI 3-9 3-20/2 EPEDFAVYYCQQYGSSPYTF 103GQGTKLEIKRADAAPTVSI 3-6 3-20/3 EPEDFAVYYCQQYGSSiFTF 104GPGTKVDIKRADAAPTVSI

Example 4: Generation and Characterization of Mice Comprising TwoHistidine-Substituted Human Light Chains Example 4.1: Engineering andGeneration of Mice Comprising Two V Kappa Segments Each Containing FourHistidine Substitutions

Histidine substitutions were introduced into the dual light chain locusas described above for Vκ1-39 and Vκ3-20 ULC mice. Briefly, the DLCsequence depicted in FIG. 23A (bottom) was subjected to site-directedmutagenesis, first modifying the Vκ1-39 sequence, and subsequentlymodifying the Vκ3-20 sequence, using primers depicted in FIG. 34. Theresultant dual light chain sequence contained Vκ1-39 segment withhistidines introduced into the germline sequence at positions 105, 106,108, and 111, Vκ3-20 segment with histidines introduced into thegermline sequence at positions 105, 106, 107, and 109, as well as allfive Jκ segments (Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5). A subsequent engineeringstep involved attachment of a 5′ arm carrying an FRT-UB-NEO-FRTcassette, and a 3′ arm carrying a mouse Igκ enhancers and constantregion. This targeting vector was electroporated into ES cellscomprising deletion of the mouse Igκ variable locus (comprising Kvariable and joining gene segments), as depicted in FIG. 35A(recombination signal sequences, RSS, are omitted in this figure).Targeted ES cells were screened by a modification of allele assay asdescribed above, using primers and probes that detected the regionsdescribed above in Tables 1, 5, 8, and 9 (specifically, 1633h2, 1635h2,neo, Jxn 1-39/3-20, mIgKd2, and mIgKp15), as well as two additional setsof primers and probes listed in Table 12 below. The sequences of thesetwo additional sets of primers and probes are included in the SequenceListing.

TABLE 12 Primers and Probes Used for ES Cell Screening Assay/ Probetype of Location Probe Forward Reverse Name  probe detected SequencePrimer Primer hVI492 GOA/ MAID 6185 AACTTA CAGCAGT GGCTCGT 1-39 FAM-(4 HIS- CTACTG CTGCAAC CCTCACA BHQ+ Jan-39 TCACCA CTGAA CATC specific)(SEQ ID (SEQ ID (SEQ ID NO: 111) NO: 112) NO: 113) hVI492 GOA/FAM-MAID 6185 TTACTGT GCAGACT AAGCTGA 3-20 BHQ+ (4 HIS- CACCAT GGAGCCTATCACTG 20-Mar CATG GAAGA TGGGAGG specific) (SEQ ID (SEQ ID TG NO: 114)NO: 115 (SEQ ID NO: 116)

A confirmed ES cell clone is then used to implant female mice to giverise to a litter of pups comprising DLC-5J light chain locus with fourhistidine modifications at each of the two present V_(L) segmentsequences, and expressing a human light chain variable domain fused witha mouse Cκ domain. Some of the same sequences as used for ES cellscreening are also used for genotyping of pups.

ES cells bearing the engineered light chain locus may be transfectedwith a construct that expresses FLP (e.g., FLPo) in order to remove theFRTed neomycin cassette introduced by the targeting construct (see FIG.35B, RSS are omitted in this figure). Optionally, the neomycin cassetteis removed by breeding to mice that express FLP recombinase (e.g., U.S.Pat. No. 6,774,279). Optionally, the neomycin cassette is retained inthe mice.

Example 4.2: Engineering and Generation of Mice Comprising Two V KappaSegments Each Containing Three Histidine Substitutions

Three histidine substitutions were introduced into each Vκ1-39 andVκ3-20 of the dual light chain mice. Briefly, the DLC sequence depictedin FIG. 23A (bottom) was subjected to site-directed mutagenesis, firstmodifying the Vκ1-39 sequence, and subsequently modifying the Vκ3-20sequence, using primers depicted in FIG. 36. The resultant dual lightchain sequence contained Vκ1-39 segment with histidines introduced intothe germline sequence at positions 106, 108, and 111, Vκ3-20 segmentwith histidines introduced into the germline sequence at positions 105,106, and 109, as well as all five Jκ segments (Jκ1, Jκ2, Jκ3, Jκ4, andJκ5). A subsequent engineering step involved attachment of a 5′ armcarrying an FRT-UB-NEO-FRT cassette, and a 3′ arm carrying a mouse Igκenhancers and constant region. This targeting vector was electroporatedinto ES cells comprising deletion of the mouse Igκ variable locus(comprising K variable and joining gene segments), as depicted in FIG.37A (RSS are omitted in this figure). Targeted ES cells were screened bya modification of allele assay as described above, using primers andprobes that detected the regions described above in Tables 1, 5, 8, and9 (specifically, 1633h2, 1635h2, neo, Jxn 1-39/3-20, mIgKd2, andmIgKp15), as well as two additional sets of primers and probes listed inTable 13 below. The sequences of these two additional sets of primersand probes are included in the Sequence Listing.

TABLE 13 Primers and Probes Used for ES Cell Screening Assay/ Probetype of Location Probe Forward Reverse Name probe detected SequencePrimer Primer hVI493 GOA/ MAID 6187 CTTACTACT CAGCAGT GGCTCGT Jan-39FAM- (3 HIS- GTCAACATAG CTGCAAC CCTCACA BHQ+ Jan-39 (SEQ ID CTGAA CATCspecific) NO: 123) (SEQ ID (SEQ ID NO: 124) NO: 125) hVI493 GOA/FAM-MAID 6187 TACTGTCAC GCAGACT AAGCTGAA 20-Mar BHQ+ (3 HIS- CATTATGGGGAGCCT TCACTGTG 20-Mar (SEQ ID GAAGA GGAGGTG specific) NO: 126) (SEQ ID(SEQ ID NO: 127) NO: 128)

A confirmed ES cell clone is then used to implant female mice to giverise to a litter of pups comprising DLC-5J light chain locus with fourhistidine modifications at each of the two present V_(L) segmentsequences, and expressing a human light chain variable domain fused witha mouse Cκ domain. Some of the same sequences as used for ES cellscreening are also used for genotyping of pups.

ES cells bearing the engineered light chain locus may be transfectedwith a construct that expresses FLP (e.g., FLPo) in order to remove theFRTed neomycin cassette introduced by the targeting construct (see FIG.37B, RSS are omitted in this figure). Optionally, the neomycin cassetteis removed by breeding to mice that express FLP recombinase (e.g., U.S.Pat. No. 6,774,279). Optionally, the neomycin cassette is retained inthe mice.

Example 4.3: Breeding of Mice Comprising a Human Histidine-SubstitutedDual Light Chains

Mice bearing an engineered human histidine-substituted dual light chainlocus are bred with mice that contain a deletion of the endogenous 2.light chain locus to generate progeny that expresses, as their onlylight chains, the engineered histidine-substituted light chains derivedfrom the dual light chain locus.

Mice bearing an engineered human histidine-substituted dual light chainlocus are bred with mice that contain a replacement of the endogenousmouse heavy chain variable locus with human heavy chain variable locus(see U.S. Pat. Nos. 6,596,541 and 8,502,018; the VELOCIMMUNE® mouse,Regeneron Pharmaceuticals, Inc.).

Example 4.4: Detection of Histidine Modifications in ImmunoglobulinLight Chains Obtained from Mice Comprising Two V Kappa Segments EachContaining Three Histidine Substitutions

V kappa amplicons from splenic B cell mRNA was prepared usingreverse-transcriptase PCR (RT-PCR) and high throughput screening.

Briefly, spleens from five heterozygous mice comprising two V kappasegments (Vκ1-39 and Vκ3-20) each containing three histidinesubstitutions (mice whose kappa locus is depicted in FIG. 35) andendogenous mouse heavy chains were harvested and homogenized in 1×PBS(Gibco) using glass slides. Cells were pelleted in a centrifuge (500×gfor 5 minutes), and red blood cells were lysed in ACK Lysis buffer(Gibco) for 3 minutes. Cells were washed with 1×PBS and filtered using a0.7 μm cell strainer. B-cells were isolated from spleen cells using MACSmagnetic positive selection for CD19 (Miltenyi Biotec). Total RNA wasisolated from pelleted B-cells using the RNeasy Plus kit (Qiagen).PolyA+ mRNA was isolated from total RNA using the Oligotex Direct mRNAmini kit (Qiagen).

Double-stranded cDNA was prepared from splenic B cell mRNA by 5′ RACEusing the SMARTer Pico cDNA Synthesis Kit (Clontech). The Clontechreverse transcriptase and dNTPs were substituted with Superscript II anddNTPs from Invitrogen. Immunoglobulin light chain repertoires wereamplified from the cDNA using primer specific for IgK constant regionand the SMARTer 5′ RACE primer (Table 14). PCR products were cleaned upusing a QIAquick PCR Purification Kit (Qiagen). A second round of PCRwas done using the same 5′ RACE primer and a nested 3′ primer specificfor the IgK constant region (Table 15). Second round PCR products werepurified using a SizeSelect E-gel system (Invitrogen). A third PCR wasperformed with primers that added 454 adapters and barcodes. Third roundPCR products were purified using Agencourt AMPure XP Beads. Purified PCRproducts were quantified by SYBR-qPCR using a KAPA LibraryQuantification Kit (KAPA Biosystems). Pooled libraries were subjected toemulsion PCR (emPCR) using the 454 GS Junior Titanium Series Lib-A emPCRKit (Roche Diagnostics) and bidirectional sequencing using Roche 454 GSJunior instrument according to the manufacturer's protocols.

TABLE 14 First Round PCR Primer SEQUENCE (SEQ ID NO) NAME 3′ mIgKAAGAAGCACACGACTGAGGCAC outer (SEQ ID NO: 129)

TABLE 15 Second Round PCR Primer NAME SEQUENCE (SEQ ID NO) 3′ mIgKGGAAGATGGATACAGTTGGTGC inner (SEQ ID NO: 130)

For bioinformatics analysis, the 454 sequence reads were sorted based onthe sample barcode perfect match and trimmed for quality. Sequences wereannotated based on alignment of rearranged Ig sequences to humangermline V and J segments database using local installation of igblast(NCBI, v2.2.25+). A sequence was marked as ambiguous and removed fromanalysis when multiple best hits with identical score were detected. Aset of perl scripts was developed to analyze results and store data inmysql database. CDR3 region of the kappa light chain was defined betweenconserved C codon and FGXG motif.

FIG. 38 represents alignments of amino acids sequence encoded by humangermline IGKV3-20 (FIG. 38A) or IGKV1-39 (FIG. 38B) sequence with aminoacid translations of exemplary Vκ sequences obtained from productivelyrearranged antibodies generated in mice comprising a histidine-modifiedDLC-5J (comprising a light chain variable locus comprising Vκ1-39 andVκ3-20 gene segments, each segment with three histidine modifications asdescribed above). The sequence reads showed that the majority ofproductively rearranged light chains retained at least one histidineintroduced into its germline CDR3. In some instances, in the majority ofall productively rearranged human light chains comprising Vκ3-20sequence that retain at least one histidine residue, all three histidinemodifications introduced into their germline CDR3 are retained (see FIG.38A). In some instances, in productively rearranged human light chainscomprising Vκ1-39 sequence that retain at least one histidine residue,about 50% of light chains retain all three histidines introduced intotheir germline CDR3 (see FIG. 38B top alignment), while about 50% oflight chains retain two out of three histidines introduced into theirgermline CDR3 (see FIG. 38B bottom alignment). In some instances,histidines at the last position of the V segment sequence may be lostdue to V-J rearrangement.

Example 5. Generation and Analysis of Mice Comprising a SingleRearranged Human Immunoglobulin Heavy Chain Nucleotide Sequence and TwoV Kappa Gene Segments

Mice comprising a rearranged heavy chain variable region nucleic acidsequence in the heavy chain locus (MAID6031; “UHC mouse”) were generatedas described above. Briefly, in the UHC mouse, all endogenous functionalheavy chain variable gene segments were deleted and replaced with asingle rearranged heavy chain variable region nucleic acid sequence thatencodes hV_(H)3-23/D/J_(H)4, which is operably linked to an endogenousheavy chain constant region nucleic acid sequence.

Mice comprising genetically engineered light chain loci containing twohuman Vκ gene segments (e.g., a human Vκ1-39 and human Vκ3-20 genesegment) and either one human Jκ segment (Jκ5; DLC-1J) or five human Jκgene segments (hJκ1-5; DLC-5J) were generated as described above.Briefly, one engineered light chain locus contains two human Vκ genesegments and five human Jκ gene segments (Jκ1-5) in unrearrangedconfiguration and is operably linked to an endogenous mouse κ constantregion sequence (MAID 1911 (DLC-5J); FIG. 19E). The other engineeredlight chain locus contains two human Vκ gene segments and one human Jκ(JO) gene segment in unrearranged configuration and is operably linkedto an endogenous mouse κ constant region sequence (MAID 1913(DLC-1J);FIG. 21D). For each of the two additional engineered light chain loci,the human gene segments were flanked 3′ with recombination signalsequences to allow for in vivo rearrangement of the human gene segmentsin B cells.

Homozygous UHC mice (MAID6031) described above were bred to homozygousDLC-5J (MAID1911) mice to produce a mouse heterozygous for the UHCallele and the DLC-5J allele. Similarly, homozygous UHC mice (MAID6031)were bred to homozygous DLC-1J (MAID1913) mice to generate a mouseheterozygous for the UHC allele and the DLC-1J allele. F1 heterozygousmice generated from these crosses were bred each other to obtain micehomozygous for each allele. The presence of the genetically modifiedalleles in the immunoglobulin heavy chain and light chain loci wasconfirmed by TAQMAN™ screening and karyotyping using specific probes andprimers described above.

Mice heterozygous for the UHC allele and the DLC-5J were bred to eachother to generate homozygotes (MAID 1912HO 6032HO; “DLC x UHC”) thatexpress immunoglobulin “light” chains mostly from the geneticallymodified locus. The MAID 1912HO 6032HO (homozygous DLC x UHC) micecomprise an insertion of the Universal Heavy Chain described herein(e.g., hV_(H)3-23/hD/hJ_(H)4) into the mouse heavy chain locus in whichall endogenouse variable heavy chain VDJ genes have been deleted andDLC-5J (hVκ1-39 hVκ3-20 hJκ1-5) the mouse kappa (κ) light chain locus inwhich all mouse Vκ and Jκ genes have been deleted.

All mice were housed and bred in specific pathogen-free conditions atRegeneron Pharmaceuticals. Three F5 VELOCIMMUNE® (MAID 1293O 1640HO(“VI3”); see U.S. Pat. No. 8,502,018, incorporated by reference herein)mice (14 weeks old, male; Background: 26.5% C57/BL6, 22.75% 129 and50.75% Balb/c) and three MAID 1912HO 6032HO F2 mice (FIG. 39; 7-8 weeksold, female; Background: 18.75 C57/BL6, 18.75% 129, and 62.5% Balb/c)were sacrificed, and spleens and bone marrow were harvested from theanimals. Bone marrow was collected from femurs by flushing with completeRPMI medium (RPMI medium supplemented with fetal calf serum, sodiumpyruvate, Hepes, 2-mercaptoethanol, non-essential amino acids, andgentamycin). Red blood cells from spleen and bone marrow preparationswere lysed with ACK lysis buffer and washed with complete RPMI medium.

Flow Cytometry

In order to examine the ability of the genetically modified homozygous“DLC x UHC” (MAID 1912HO 6032HO) mice described herein to produceantibodies derived from the genetically modified alleles (e.g., from theallele that contains a single copy of the rearranged V_(H)3-23/D/J_(H)4in the heavy chain locus and the allele that contains two human Vκ fivehuman Jκ genes in the light chain locus), fluorescence-activated cellsorting (FACS) analysis was performed as in Example 3.

In the splenic compartment, MAID 1912HO 6032HO mice demonstrated CD19+ Bcell numbers and mature B cell numbers that were substantially the sameas the numbers observed in VELOCIMMUNE® (VI3) mice (FIGS. 40A-40B),which serve as a control for the specific effects observed in MAID1912HO 6032HO mice relative to mice with other genetic modifications intheir immunoglobulin loci; also, the humoral immune system ofVELOCIMMUNE® mice functions like that of wild type mice (supra). TheMAID 1912HO 6032HO mice demonstrated a 2-fold increase in immature Bcell numbers in the spleen compared to VI3 mice (FIGS. 40A-40B). TheMAID 1912HO 6032HO mice were also substantially similar to VI3 mice withrespect to kappa and gamma light chain usage (FIGS. 41A-41B). MAID1912HO 6032HO (DLC x UHC) mice also demonstrated increased surface IgMon splenic B cells (i.e., more IgM surface expression per cell) ascompared to VI3 mice (FIG. 42).

Also, the MAID 1912HO 6032HO (DLC x UHC) mice demonstrated alteredperipheral B cell development as progression of B cells through variousstages in the splenic compartment (e.g., immature, mature, T1, T2 T3,marginal zone precursor, marginal zone, follicular-1, follicular-11,etc.) occurred in a different manner than observed in VI3 mice (FIG.43A). Specifically, the MAID 1912HO 6032HO (DLC x UHC) mice demonstratedmore immature, T1 and marginal zone (MZ) B cells in the spleniccompartment as compared to VI3 mice. The numbers of follicular-1 andfollicular-11 cells in the MAID 1912HO 6032HO (DLC x UHC) mice weresubstantially the same as observed in VI3 mice (FIG. 43B).

In the bone marrow compartment, MAID 1912HO 6032HO (DLC x UHC) micedemonstrated similar numbers of CD19+ B cells compare to VI3 micecontrols (FIGS. 44A-44B). However, the MAID 1912HO 6032HO (DLC x UHC)mice demonstrated about 25-fold fewer pro-B cells in the bone marrow ascompared to VI3 mice (FIGS. 45A-45B). The MAID 1912HO 6032HO (DLC x UHC)mice also demonstrated about 2-fold less immature B cells and 2-foldless mature B cells in the bone marrow compared to VI3 mice (FIGS.46A-46B). Also, the MAID 1912HO 6032HO (DLC x UHC) mice demonstrated apreference (2-fold increase) for lambda expression compared to VI3 mice(FIG. 47).

Immunization Studies

Five WT (75% C57BL6/25% 129 background) and seven F2 MAID1912HO 6031 HET(homozygous DLC x heterozygous UHC) mice were immunized in the footpadwith 0.025 ml of a mixture containing 2.35 μg of an antigen X, 10 μg CpGoligonucleotide (ODN 1826, InvivoGen, cat #tlrl-1826), and 25 μgAluminum Phosphate Gel Adjuvant (Brenntag cat #7784-30-7). Mice wereboosted six times with the same dosage. On days 0, 15 and 23 postprimary immunization, blood was collected from anaesthetized mice usinga retro-orbital bleed into BD serum separator tubes (BD, cat #365956),and serum was collected as per manufacturer's directions. A second roundof immunization was performed as above five weeks after the first roundof immunization.

To measure the levels of antigen-specific IgG antibodies, ELISAs wereperformed as in Example 3. As shown in FIG. 48, the genetically modifiedmice, which are heterozygous with respect to the targeted allelecontaining the rearranged V_(H)3-23/D/J_(H)4 nucleotide sequence andhomozygous with respect to the targeted allele containing DLC-5J, wereable to produce antigen-specific IgG antibodies at levels comparable tothose produced by wild type mice at both 23 days and 5 weeks after theprimary immunization. The MAID1912HO 6031 HET (homozygous DLC xheterozygous UHC) mice were also able to produce antigen-specific IgGantibodies at levels comparable to those produced by wild type miceafter the 2^(nd) round of immunization.

Thus, these mice produce antibodies comprising a reverse chimeric lightchain (human light chain variable domain and mouse Cκ) derived from arearrangement of one of the two human V_(L) gene segments (Vκ1-39 orVκ3-20 gene segments) and human Jκ segments and a reverse chimeric heavychain (human heavy chain variable domain and mouse C_(H)) derived from asingle rearranged human heavy chain variable gene segment. Reversechimeric antibodies (i.e., antibodies comprised of these reversechimeric chains) are obtained upon immunization with an antigen ofinterest.

Example 6. Generation and Analysis of Mice Comprising a SingleRearranged Human Immunoglobulin Heavy Chain Nucleotide Sequence and TwoV Kappa Gene Segments Containing Three Histidine Substitutions

Similarly, mice bearing an engineered human light chain locus comprisinga histidine-modified dual light chain (e.g., mice comprising two humanV_(L) gene segments with histidine modifications described herein above)are bred with mice that contain a replacement of the endogenous mouseheavy chain variable locus with universal human heavy chain locus (locuscomprising a single rearranged human heavy chain variable domain asdescribed herein above). Thus, these mice produce antibodies comprisinga reverse chimeric light chain (human light chain variable domain andmouse Cκ) derived from a rearrangement of one of the twohistidine-modified human V_(L) gene segments (Vκ1-39 or Vκ3-20 genesegments) and human Jκ segments and a reverse chimeric heavy chain(human heavy chain variable domain and mouse CH) derived from a singlerearranged human heavy chain variable domain. Reverse chimericantibodies are obtained upon immunization with an antigen of interest.pH-dependent human antibodies generated in such mice are identifiedusing antibody isolation and screening methods known in the art ordescribed above.

Variable light and heavy chain region nucleotide sequences of B cellsexpressing the antibodies are identified, and fully human light andheavy chains are made by fusion of the variable light and heavy chainregion nucleotide sequences to human C_(L) and C_(H) nucleotidesequences, respectively. Light chains of interest, e.g., light chainsthat bind to the antigen of interest (e.g., light chains from antibodiesthat also demonstrate pH-dependent antigen properties using a variety ofassays known in the art, e.g., BIACORE™ assay) are co-expressed in asuitable expression system with heavy chains derived from otherantibodies, e.g., heavy chains derived from antibodies that compriselight chains derived from the same V_(L) gene segment as that in thelight chain of interest (e.g., Vκ1-39 or Vκ3-20), and the reconstitutedantibody is tested for its ability to retain antigen-binding andpH-dependent antigen-binding properties.

Example 7. Construction of Mice Comprising an Immunoglobulin Light ChainLocus Containing a Rearranged Heavy Chain VDJ Sequence

Mice comprising a rearranged heavy chain variable region nucleic acidsequence in the kappa light chain locus (MAID6079; “UHC on kappa mouse”)were generated by similar methods to those described above for targetingthe heavy chain locus. Briefly, in the UHC on kappa mouse, allendogenous functional light chain kappa variable Vκ and Jκ gene segmentswere deleted and replaced with a single rearranged heavy chain variableregion nucleic acid sequence that encodes hV_(H)3-23/D/J_(H)4, which isoperably linked to an endogenous light chain constant region nucleicacid sequence. The final targeting construct for the creation of agenomic locus containing a rearranged human heavy chain variable domainsequence contains, from 5′ to 3′, (1) a 5′ homology arm containing about22500 bp of a mouse genomic sequence upstream of the endogenous Ig lightchain locus; (2) a 5′ FRT site; (3) a neomycin cassette; (4) a 3′ FRTsite, (5) 2239 bp of hV_(H)3-23 promoter (SEQ ID NO: 139); (6) arearranged human immunoglobulin heavy chain nucleotide sequence(hV_(H)3-23/D/J_(H)4; SEQ ID NO: 136); (7) an hJ_(H)4 intron (SEQ ID NO:140); and (8) a 3′ homology arm containing about 75000 bp of a mousegenomic sequence downstream of the mouse JL gene segments. Heterozygousmice bearing the modification were bred to each other to generatehomozygotes (MAID 6079HO) that are capable of making immunoglobulin“light” chains only from the genetically modified locus. The MAID 6079HO(homozygous UHC on kappa) mice comprise an insertion of the UniversalHeavy Chain described herein (e.g., hV_(H)3-23/hD/hJ_(H)4) into themouse kappa (κ) light chain locus in which all mouse Vκ and Jκ geneshave been deleted.

All mice were housed and bred in specific pathogen-free conditions atRegeneron Pharmaceuticals. Four MAID 6079HO F1 mice (FIG. 49; 7-12.5weeks old, male and female) and four MAID 6079 F1 wild type littermatecontrol mice (7-12.5 weeks old, male and female) were sacrificed, andspleens and bone marrow were harvested from the animals. Bone marrow wascollected from femurs by flushing with complete RPMI medium (RPMI mediumsupplemented with fetal calf serum, sodium pyruvate, Hepes,2-mercaptoethanol, non-essential amino acids, and gentamycin). Red bloodcells from spleen and bone marrow preparations were lysed with ACK lysisbuffer and washed with complete RPMI medium.

Flow Cytometry

In order to examine the ability of the genetically modified homozygous“UHC on kappa mouse” (MAID 6079HO) mice described herein to produceantibodies derived from the genetically modified allele (e.g., from theallele that contains a single copy of the rearranged V_(H)3-23/D/J_(H)4in a kappa light chain locus), fluorescence-activated cell sorting(FACS) analysis was performed as in Example 3.

The MAID 6079HO mice demonstrated numbers of pro- and pre-B cells in thebone marrow compartment that are substantially the same as observed inwild type littermates (FIGS. 50A-50B). In contrast, they demonstratedlower numbers of immature and mature B cells in the bone marrowcompartment compared to wild type littermates (FIGS. 51A-51C). In fact,the mice had 2-fold less immature B cells, and almost 4-fold less matureB cells. The MAID 6079HO mice almost exclusively used lambda light chainsequences in immature and mature B cells in the bone marrow (FIG. 52).

In the splenic compartment, MAID 6079HO mice demonstrated fewer mature Bcells compared to wild type littermates (FIGS. 53A-53B). Similar to whatwas observed in the bone marrow, MAID 6079HO mice almost exclusivelyused lambda light chain sequences in the splenic compartment (FIGS.54A-54B). They also demonstrated fewer immature cells, an increase inmarginal zone B cells and a decrease in follicular B cells compared towild type littermates (FIG. 55).

Example 8. Generation and Analysis of Mice Comprising an ImmunoglobulinLight Chain Locus Containing a Rearranged Heavy Chain VDJ Sequence andan Immunoglobulin Heavy Chain Locus Containing a Human Light ChainVariable Domain Sequence

Mice homozygous for a rearranged heavy chain variable region nucleicacid sequence in the light chain locus (MAID 6079HO; homozygous “UHC onkappa mouse”) were generated as described above. These mice were crossedto mice homozygous (MAID 1994HO) for a kappa light chain variable regionnucleic acid sequence in a heavy chain locus (kappa on heavy (“KoH”)mouse). The MAID 1994 homozygous KoH mice comprise 40 human Vκ genes andall human Jκ genes, with long IGCR and mouse ADAM6, inserted into amouse Ig heavy chain constant chain locus (i.e., a deleted mouse Igheavy chain locus)). KoH have been described previously; see, e.g., U.S.pre-grant publication 2012/0096572, incorporated herein by reference.

All mice were housed and bred in specific pathogen-free conditions atRegeneron Pharmaceuticals. Two VELOCIMMUNE® (MAID 1242HO 1640HO (“VI3”);see U.S. Pat. No. 8,502,018, incorporated by reference herein) mice (15weeks old, female n=2; Background: 28% C57/BL6, 13% 129 and 59% Balb/c),four MAID 1994HO 6079HO F2 mice (FIG. 56; 13-14 weeks old, male n=2;Background: 25% C57/BL6, 25% 129, and 50% Balb/c), and MAID 6079 wildtype littermate control mice were sacrificed, and spleens and bonemarrow were harvested from the animals. Bone marrow was collected fromfemurs by flushing with complete RPMI medium (RPMI medium supplementedwith fetal calf serum, sodium pyruvate, Hepes, 2-mercaptoethanol,non-essential amino acids, and gentamycin). Red blood cells from spleenand bone marrow preparations were lysed with ACK lysis buffer and washedwith complete RPMI medium.

Flow Cytometry

In order to examine the ability of the genetically modified homozygous“KoH x UHC on kappa” (MAID 1994HO 6079HO) mice described herein toproduce antibodies derived from the genetically modified alleles (e.g.,from the allele that contains a single copy of the rearrangedV_(H)3-23/D/J_(H)4 and the allele that contains a kappa light chainvariable region nucleic acid sequence in a heavy chain locus),fluorescence-activated cell sorting (FACS) analysis was performed as inExample 3.

MAID 1994HO 6079HO mice demonstrated lower CD19+ and pre-B cellfrequencies in the bone marrow compartment compared to VI3 mice (FIG.57A). Specifically, the MAID 19940 6079HO mice demonstrated about a2-fold lower CD19+ and pre-B cell numbers in the bone marrow compared toVI3 mice (FIG. 57B). Additionally, the MAID 1994HO 6079HO micedemonstrated about 3-fold less immature B cells in the bone marrowcompartment relative to VI3 mice (FIGS. 58A and 58B). It was also foundthat B cells from the MAID 1994HO 6079HO mice essentially lackexpression of lambda light chain in the bone marrow (FIG. 59).

MAID1994HO 6079HO mice demonstrated a lower frequency of B cells in thesplenic compartment. Specifically, MAID 1994HO 6079HO mice had fewersplenic B cells (about 2-fold less) and mature B cells (about 3-foldless) numbers relative to VI3 mice (FIGS. 60A-60B. They againdemonstrated a lack expression of lambda light chain as compared to VI3mice (FIG. 61).

Considering peripheral B cell development in the spleen, the FACSanalysis indicated that MAID1994HO 6079HO mice have an increasedfrequency of cells in T1 phase in the spleen than VI3 mice (FIG. 62).

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areencompassed by the following claims.

Entire contents of all non-patent documents, patent applications andpatents cited throughout this application are incorporated by referenceherein in their entirety.

While the described invention has been described with reference toparticular aspects and embodiments thereof, those skilled in the artunderstand that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adopt aparticular situation, material, composition of matter, process, processstep or steps, to the objective spirit and scope of the describedinvention. All such modifications are within the scope of the claimsappended hereto.

1.-106. (canceled)
 107. A nucleotide molecule comprising a rearrangedhuman immunoglobulin heavy chain variable region nucleotide sequencethat comprises a human germline heavy chain V_(H) gene segment and/or ahuman germline heavy chain J_(H) gene segment.
 108. The nucleotidemolecule of claim 107, wherein (i) the human germline heavy chain V_(H)gene segment is selected from the group consisting of V_(H)1-2,V_(H)1-3, V_(H)1-8, V_(H)1-18, V_(H)1-24, V_(H)1-45, V_(H)1-46,V_(H)1-58, V_(H)1-69, V_(H)2-5, V_(H)2-26, V_(H)2-70, V_(H)3-7,V_(H)3-9, V_(H)3-11, V_(H)3-13, V_(H)3-15, V_(H)3-16, V_(H)3-20, V_(H)3-21, V_(H)3-23, V_(H)3-30, V_(H)3-30-3, V_(H) 3-30-5, V_(H)3-33,V_(H)3-35, V_(H)3-38, V_(H)3-43, V_(H)3-48, V_(H)3-49, V_(H)3-53,V_(H)3-64, V_(H)3-66, V_(H)3-72, V_(H)3-73, V_(H)3-74, V_(H)4-4,V_(H)4-28, V_(H)4-30-1, V_(H)4-30-2, V_(H)4-30-4, V_(H)4-31, V_(H)4-34,V_(H)4-39, V_(H)4-59, V_(H)4-61, V_(H)5-51, V_(H)6-1, V_(H)7-4-1,V_(H)7-81, and any polymorphic variant thereof. and/or (ii) the humangermline heavy chain J_(H) gene segment is selected from the groupconsisting of J_(H)1, J_(H)2, J_(H)3, J_(H)4, J_(H)5, J_(H)6, and anypolymorphic variant thereof
 109. The nucleotide molecule of claim 107,wherein the rearranged human immunoglobulin heavy chain variable regionsequence comprises a human germline heavy chain V_(H) gene segment and ahuman germline heavy chain J_(H) gene segment.
 110. The nucleotidemolecule of 107, wherein the rearranged human immunoglobulin heavy chainvariable region sequence encodes the sequence of V_(H)3-23/X₁X2/J_(H),wherein X₁ is any amino acid, and X2 is any amino acid.
 111. Thenucleotide molecule of claim 110, wherein X₁ is Gly and X₂ is Tyr. 112.The nucleotide molecule of claim 111, wherein J_(H) is selected from thegroup consisting of a human germline J_(H)1 gene segment, a humangermline J_(H)2 gene segment, a human germline J_(H)3 gene segment, ahuman germline J_(H)4 gene segment, a human germline J_(H)5 genesegment, a human germline J_(H)6 gene segment, and a polymorphic variantthereof.
 113. The nucleotide molecule of claim 107, wherein therearranged human immunoglobulin heavy chain variable region sequenceencodes the sequence of human V_(H)3-23/GY/J_(H)4-4 (SEQ ID NO:137).114. The nucleotide molecule of claim 107, comprising the rearrangedhuman immunoglobulin heavy chain variable region nucleotide sequenceoperably linked to an immunoglobulin heavy chain constant regionnucleotide sequence.
 115. The nucleotide molecule of claim 114, whereinthe heavy chain constant region gene sequence is selected from a C_(H)1,a hinge, a C_(H)2, a C_(H)3, and a combination thereof.
 116. Thenucleotide molecule of claim 114, wherein the immunoglobulin heavy chainconstant region nucleotide sequence is a human immunoglobulin heavychain constant region nucleotide sequence.
 117. An immunoglobulinpolypeptide comprising an amino acid sequence encoded by the nucleotidemolecule of claim
 117. 118. A host cell comprising the nucleotidemolecule of claim
 107. 119. The host cell of claim 118, furthercomprising a nucleic acid sequence that encodes a human light chainvariable domain.
 120. The host cell of claim 119, wherein the nucleicacid sequence that encodes a human light chain variable domain isoperably linked to an immunoglobulin light chain constant region genesequence.
 121. The host cell of claim 120, wherein the immunoglobulinlight chain constant region gene sequence is a human immunoglobulinlight chain constant region gene sequence.
 122. The host cell of claim119, further comprising (i) an immunoglobulin heavy chain variabledomain encoded by the immunoglobulin heavy chain variable regionnucleotide sequence that comprises a human germline heavy chain V_(H)gene segment and/or a human germline heavy chain J_(H) gene segment and(ii) the immunoglobulin light chain variable domain.
 123. Anantigen-binding protein expressed by the host cell of claim 122, whereinthe antigen-binding protein comprises (i) the immunoglobulin heavy chainvariable domain, and (ii) the immunoglobulin light chain variabledomain.